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• First Law of Thermodynamics o Enthalpy o Reversible and Irreversible Reactions

• Second Law of Thermodynamics and Entropy • Standard State Conditions for Biological Reactions • Coupled Reactions

Return to Medical Biochemistry Page

First Law of Thermodynamics

Stated simply; The total energy of the universe does not change. This does not mean that the form of the energy cannot change. Indeed, chemical energies of a molecule can be converted to thermal, electrical or mechanical energies. The internal energy of a system can change only by work or heat exchanges. From this the change in the free energy of a system can be shown by the following equation:

∆E = q - w Eqn. 1 When q is negative heat has flowed from the system and when q is positive heat has been absorbed by the system. Conversely when w is negative work has been done on the system by the surrounding and when positive, work has been done by the system on the surroundings. In a reaction carried out at constant volume no work will be done on or by the system, only heat will be transferred from the system to the surroundings. The end result is that:

∆E = q Eqn. 2 When the same reaction is performed at constant pressure the reaction vessel will do work on the surroundings. In this case:

∆E = q - w Eqn. 3 where w = P∆V Eqn. 4

When the initial and final temperatures are essentially equal (e.g. in the case of biological systems):

∆V = ∆n[RT/P] Eqn. 5 therefore, w = ∆nRT Eqn. 6

by rearrangement of equation 3 and incorporation of the statements in equations 4-6, one can calculate the amount of heat released under constant pressure:

q = ∆E + w = ∆E + P∆V = ∆E + ∆nRT Eqn. 7

In equation 7 ∆n is the change in moles of gas per mole of substance oxidized (or reacted), R is the gas constant and T is absolute temperature. back to the top

Enthalpy

Since all biological reactions take place at constant pressure and temperature the state function of reactions defined to account for the heat evolved (or absorbed) by a system is enthalpy given the symbol, H. The changes in enthalpy are related to changes in free energy by the following equation:

∆H = ∆E + P∆V Eqn. 8 Equation 8 is in this form because we are addressing the constant pressure situation. In the biological setting most all reaction occur in a large excess of fluid, therefore, essentially no gases are formed during the course of the reaction. This means that the value ∆V, is extremely small and thus the product P∆V is very small as well. The values ∆E and ∆H are very nearly equivalent in biological reactions Stated above was the fact that state functions, like ∆H and ∆E, do not depend on the path taken during a reaction. These functions pertain only to the differences between the initial and final states of a reaction. However, heat (q) and work (w) are not state functions and their values are affects by the pathway taken. back to the top

Reversible and Irreversible Reactions

In an idealized irreversible reaction such as one done by expanding an ideal gas against zero pressure, no work will be done by or on the system so the:

w = 0 Eqn. 9 In the case of an ideal gas (whose molecules do not interact) there will be no change in internal energy either so:

∆E = 0 Eqn. 10

since ∆E = q - w, in this irreversible reaction q = 0 also.

In a reversible reaction involving an ideal gas, ∆E still will equal zero, however, the pressure will be changing continuously and work (w) is a funtion of P, work done must be determined over the entire course of the reaction. This result in the following mathematical reduction:

w = RTln[V2/V1] Eqn. 11 Since in this situation ∆E = 0, q = w. This demonstrates that some of the heat of the surroundings has to be absorbed by the system in order to perform the work of changing the system volume. Reversible reactions differ from irreversible in that the former always proceeds infinitely slowly through a series of intermediate steps in which the system is always in the equilibrium state. Whereas, in the irreversible reaction no

equilibrium states are encountered. Irreversible reactions are also spontaneous or favorable processes. Thermodynamic calculations do not give information as to the rates of reaction only whether they are favorable or not. back to the top

Second Law of Thermodynamics: Entropy

The second law of thermodynamics states that the universe (i.e. all systems) tend to the greatest degree of randomization. This concept is defined by the term entropy, S.

S = klnW Eqn. 12

where k = Boltzmann constant (the gas constant, R, divided by Avagadros' number) and W = the number of substrates. For an isothermal reversible reaction the change in entropy can be reduced to the term:

∆S = ∆H/T Eqn. 13 Whereas, enthalpy is a term whose value is largely dependent upon electronic internal energies, entropy values are dependent upon translational, vibrational and rotational internal energies. Entropy also differs from enthalpy in that the values of enthalpy that indicate favored reactions are negative and the values of entropy are positive. Together the terms enthalpy and entropy demonstrate that a system tends toward the highest entropy and the lowest enthalpy. In order to effectively evaluate the course (spontaneity or lack there of) of a reaction and taking into account both the first and second laws of thermodynamics, Josiah Gibbs defined the term, free energy. Free energy:

∆G = ∆H - T∆S Eqn. 14 Free energy is a valuable concept because it allows one to determine whether a reaction will proceed and allows one to calculate the equilibrium constant of the reaction which defines the extent to which a reaction can proceed. The discussion above indicated that a decrease in energy, a negative∆H, and an increase in entropy, a positive ∆S, are indicative of favorable reactions. These terms would, therefore, make ∆G a negative value. Reactions with negative ∆G values are termed exergonic and those with positive ∆G values endergonic. However, when a system is at equilibrium:

∆G = 0 Eqn. 15 Gibb's free energy calculations allows one to determine whether a given reaction will be thermodynamically favorable. The sign of ∆G states that a reaction as written or its reverse process is the favorable step. If ∆G is negative then the forward reaction is favored and visa versa for ∆G values that are calculated to be positive. back to the top

Standard State Conditions in Biological Reactions To effectively interpret the course of a reaction in the presence of a mixture of components, such as in the cell, one needs to account for the free energies of

the contributing components. This is accomplished by calculating total free energy which is comprised of the individual free energies. In order to carry out these calculations one needs to have a reference state from which to calculate free energies. This reference state, termed the Standard State, is chosen to be the condition where each component in a reaction is at 1M. Standard state free energies are given the symbol:

Go

The partial molar free energy of any component of the reaction is related to the standard free energy by the following:

G = Go + RTln[X] Eqn. 16 From equation 16 one can see that when the component X, or any other component, is at 1M the ln[1] term will become zero and:

G = Go Eqn. 17 The utility of free energy calculations can be demonstrated in a consideration of the diffusion of a substance across a membrane. The calculation needs to take into account the changes in the concentration of the substance on either side of the membrane. This means that there will be a ∆G term for both chambers and, therefore, the total free energy change is the sum of the ∆G values for each chamber:

∆G = ∆G1 + ∆G2 = RTln{[A]2/[A]1}Eqn. 18 Equation 18 tells one that if [A]2 is less than [A]1 the value of ∆G will be negative and transfer from region 1 to 2 is favored. Conversely if [A]2 is greater than [A]1 ∆G will be positive and transfer from region 1 to 2 is not favorable, the reverse direction is. One can expand upon this theme when dealing with chemical reactions. It is apparent from the derivation of ∆G values for a given reaction that one can utilize this value to determine the equilibrium constant, Keq. As for the example above dealing with transport across a membrane, calculation of the total free energy of a reaction includes the free energies of the reactants and products:

∆G = G(products) - G(reactants) Eqn. 19 Since this calculation involves partial molar free energies the ∆Go terms of all the reactants and products are included. The end result of the reduction of all the terms in the equation is:

∆G =∆Go + RTln{[C][D]/[A][B]} Eqn. 20 When equation 20 is used for a reaction that is at equilibrium the concentration values of A, B, C and D will all be equilibrium concentrations and, therefore, will be equal to Keq. Also, when at equilibrium ∆G = 0. Therefore:

0 =∆Go + RTlnKeq Eqn. 21

Keq = e-{∆Go/RT} Eqn. 22

This demonstrates the relationship between the free energy values and the equilibrium constants for any reaction. back to the top

Coupled Reactions

Two or more reactions in a cell sometimes can be coupled so that thermodynamically unfavorable reactions and favorable reactions are combined to drive the overall process in the favorable direction. In this circumstance the overall free energy is the sum of individual free energies of each reaction. This process of coupling reactions is carried out at all levels within cells. The predominant form of coupling is the use of compounds with high energy to drive unfavorable reactions. The predominant form of high energy compounds in the cell are those which contain phosphate. Hydrolysis of the phosphate group can yield free energies in the range of -10 to -62 kJ/mol. These molecules contain energy in the phosphate bonds due to:

• 1. Resonance stabilization of the phosphate products • 2. Increased hydration of the products • 3. Electrostatic repulsion of the products • 4. Resonance stabilization of products • 5. Proton release in buffered solutions

The latter phenomenon indicates that the pH of the solution a reaction is performed in will influence the equilibrium of the reaction. To account for the fact that all cellular reactions take place in an aqueous environment and that the [H2O] and [H+] are essentially constant these terms in the free energy calculation have been incorporated into a free energy term identified as:

∆Go' =∆Go + RTln{[H+]/[H2O]} Eqn. 23

Incorporation of equation 23 into a free energy calculation for any reaction in the cell yields:

∆G =∆Go' + RTln{[products]/[reactants]} Eqn. 24 back to the top


Michael W. King, Ph.D / IU School of Medicine / [email protected]

Last modified: Tuesday, 12-Aug-2003 20:06:22 EST

• Chemistry of Amino Acids

• Amino Acid Classifications • Acid-Base Properties • Functional Significance of R-Groups • Optical Properties • The Peptide Bond


Chemical Nature of the Amino Acids

All peptides and polypeptides are polymers of alpha-amino acids. There are 20 α-amino acids that are relevant to the make-up of mammalian proteins (see below). Several other amino acids are found in the body free or in combined states (i.e. not associated with peptides or proteins). These non-protein associated amino acids perform specialized functions. Several of the amino acids found in proteins also serve functions distinct from the formation of peptides and proteins, e.g., tyrosine in the formation of thyroid hormones or glutamate acting as a neurotransmitter. The α-amino acids in peptides and proteins (excluding proline) consist of a carboxylic acid (-COOH) and an amino (-NH2) functional group attached to the same tetrahedral carbon atom. This carbon is the α-carbon. Distinct R-groups, that distinguish one amino acid from another, also are attached to the alpha-carbon (except in the case of glycine where the R-group is hydrogen). The fourth substitution on the tetrahedral α-carbon of amino acids is hydrogen.

Table of αααα-Amino Acids Found in Proteins

Amino Acid

Symbol Structure*

pK1 (COO

H)

pK2 (NH

2)

pK R Grou

p

Amino Acids with Aliphatic R-Groups

Glycine Gly - G

2.4 9.8

Alanine Ala - A

2.4 9.9

Valine Val - V

2.2 9.7

Leucine Leu - L

2.3 9.7

Isoleucine Ile - I 2.3 9.8

Non-Aromatic Amino Acids with Hydroxyl R-Groups

Serine Ser - S

2.2 9.2 ~13

Threonine Thr - T

2.1 9.1 ~13

Amino Acids with Sulfur-Containing R-Groups

Cysteine Cys - C

1.9 10.8 8.3

Methionine Met-M

2.1 9.3

Acidic Amino Acids and their Amides

Aspartic Acid

Asp - D

2.0 9.9 3.9

Asparagine Asn - N

2.1 8.8

Glutamic Acid

Glu - E 2.1 9.5 4.1

Glutamine Gln - Q 2.2 9.1

Basic Amino Acids

Arginine Arg - R 1.8 9.0 12.5

Lysine Lys - K

2.2 9.2 10.8

Histidine His - H

1.8 9.2 6.0

Amino Acids with Aromatic Rings

Phenylalanine

Phe - F

2.2 9.2

Tyrosine Tyr - Y 2.2 9.1 10.1

Tryptophan Trp-W 2.4 9.4

Imino Acids

Proline Pro - P

2.0 10.6

*Backbone of the amino acids is red, R-groups are black back to the top

Amino Acid Classifications

Each of the 20 α-amino acids found in proteins can be distinguished by the R-group substitution on the α-carbon atom. There are two broad classes of amino

acids based upon whether the R-group is hydrophobic or hydrophilic. The hydrophobic amino acids tend to repel the aqueous environment and,

therefore, reside predominantly in the interior of proteins. This class of amino

acids does not ionize nor participate in the formation of H-bonds. The hydrophilic amino acids tend to interact with the aqeuous environment, are often involved in the formation of H-bonds and are predominantly found on the exterior surfaces

proteins or in the reactive centers of enzymes. back to the top

Acid-Base Properties of the Amino Acids

The α-COOH and α-NH2 groups in amino acids are capable of ionizing (as are the acidic and basic R-groups of the amino acids). As a result of their ionizability

the following ionic equilibrium reactions may be written: R-COOH <--------> R-COO- + H+

R-NH3+ <---------> R-NH2 + H+

The equilibrium reactions, as written, demonstrate that amino acids contain at least two weakly acidic groups. However, the carboxyl group is a far stronger

acid than the amino group. At physiological pH (around 7.4) the carboxyl group will be unprotonated and the amino group will be protonated. An amino acid with

no ionizable R-group would be electrically neutral at this pH. This species is termed a zwitterion.

Like typical organic acids, the acidic strength of the carboxyl, amino and ionizable R-groups in amino acids can be defined by the association constant, Ka

or more commonly the negative logrithm of Ka, the pKa. The net charge (the algebraic sum of all the charged groups present) of any amino acid, peptide or

protein, will depend upon the pH of the surrounding aqueous environment. As the pH of a solution of an amino acid or protein changes so too does the net charge.

This phenomenon can be observed during the titration of any amino acid or protein. When the net charge of an amino acid or protein is zero the pH will be

equivalent to the isoelectric point: pI.

Titration curve for Alanine

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Functional Significance of Amino Acid R-Groups

In solution it is the nature of the amino acid R-groups that dictate structure-function relationships of peptides and proteins. The hydrophobic amino acids will generally be encountered in the interior of proteins shielded from direct contact with water. Conversely, the hydrophilic amino acids are generally found on the

exterior of proteins as well as in the active centers of enzymatically active proteins. Indeed, it is the very nature of certain amino acid R-groups that allow

enzyme reactions to occur. The imidazole ring of histidine allows it to act as either a proton donor or acceptor

at physiological pH. Hence, it is frequently found in the reactive center of enzymes. Equally important is the ability of histidines in hemoglobin to buffer the

H+ ions from carbonic acid ionization in red blood cells. It is this property of hemoglobin that allows it to exchange O2 and CO2 at the tissues or lungs,

respectively.

The primary alcohol of serine and threonine as well as the thiol (-SH) of cysteine allow these amino acids to act as nucleophiles during enzymatic catalysis. Additionally, the thiol of cysteine is able to form a disulfide bond with other

cysteines: Cysteine-SH + HS-Cysteine <--------> Cysteine-S-S-Cysteine

This simple disulfide is identified as cystine. The formation of disulfide bonds between cysteines present within proteins is important to the formation of active

structural domains in a large number of proteins. Disulfide bonding between cysteines in different polypeptide chains of oligomeric proteins plays a crucial

role in ordering the structure of complex proteins, e.g. the insulin receptor. back to the top

Optical Properties of the Amino Acids

A tetrahedral carbon atom with 4 distinct constituents is said to be chiral. The one amino acid not exhibiting chirality is glycine since its '"R-group" is a hydrogen atom. Chirality describes the handedness of a molecule that is observable by the

ability of a molecule to rotate the plane of polarized light either to the right (dextrorotatory) or to the left (levorotatory). All of the amino acids in proteins exhibit the same absolute steric configuration as L-glyceraldehyde. Therefore,

they are all L-α-amino acids. D-amino acids are never found in proteins, although they exist in nature. D-amino acids are often found in polypetide antibiotics.

The aromatic R-groups in amino acids absorb ultraviolet light with an absorbance maximum in the range of 280nm. The ability of proteins to absorb ultraviolet light is predominantly due to the presence of the tryptophan which strongly absorbs

ultraviolet light. back to the top

The Peptide Bond

Peptide bond formation is a condensation reaction leading to the polymerization of amino acids into peptides and proteins. Peptides are small consisting of few

amino acids. A number of hormones and neurotransmitters are peptides. Additionally, several antibiotics and antitumor agents are peptides. Proteins are

polypeptides of greatly divergent length. The simplest peptide, a dipeptide, contains a single peptide bond formed by the condensation of the carboxyl group

of one amino acid with the amino group of the second with the concomitant elimination of water. The presence of the carbonyl group in a peptide bond allows

electron resonance stabilization to occur such that the peptide bond exhibits rigidity not unlike the typical -C=C- double bond. The peptide bond is, therefore,

said to have partial double-bond character.

Resonance stabilization forms of the peptide bond

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Return to Basic Chemistry of Biomolecules




• Introduction to Carbohydrates • Carbohydrate Nomenclature • Monosaccharides • Disaccharides • Polysaccharides • Glycogen • Starch


Introduction

Carbohydrates are carbon compounds that contain large quantities of hydroxyl groups. The simplest carbohydrates also contain either an aldehyde moiety (these are termed polyhydroxyaldehydes) or a ketone moiety (polyhydroxyketones). All carbohydrates can be classified as either monosaccharides, oligosaccharides or polysaccharides. Anywhere from two to ten monosaccharide units, linked by glycosidic bonds, make up an

oligosaccharide. Polysaccharides are much larger, containing hundreds of monosaccharide units. The presence of the hydroxyl groups allows carbohydrates to interact with the aqueous environment and to participate in hydrogen bonding, both within and between chains. Derivatives of the carbohydrates can contain nitrogens, phosphates and sulfur compounds. Carbohydrates also can combine with lipid to form glycolipids or with protein to form glycoproteins. back to the top

Carbohydrate Nomenclature

The predominant carbohydrates encountered in the body are structurally related to the aldotriose glyceraldehyde and to the ketotriose dihydroxyacetone. All carbohydrates contain at least one asymmetrical (chiral) carbon and are, therefore, optically active. In addition, carbohydrates can exist in either of two conformations, as determined by the orientation of the hydroxyl group about the asymmetric carbon farthest from the carbonyl. With a few exceptions, those carbohydrates that are of physiological significance exist in the D-conformation. The mirror-image conformations, called enantiomers, are in the L-conformation.

Structures of Glyceraldehyde Enantiomers

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Monosaccharides The monosaccharides commonly found in humans are classified according to the

number of carbons they contain in their backbone structures. The major monosaccharides contain four to six carbon atoms.

Carbohydrate Classifications

# Carbons

Category Name

Relevant examples

3 Triose Glyceraldehyde, Dihydroxyacetone

4 Tetrose Erythrose

5 Pentose Ribose, Ribulose, Xylulose

6 Hexose Glucose, Galactose, Mannose, Fructose

7 Heptose Sedoheptulose

9 Nonose Neuraminic acid also called sialic acid

The aldehyde and ketone moieties of the carbohydrates with five and six carbons

will spontaneously react with alcohol groups present in neighboring carbons to produce intramolecular hemiacetals or hemiketals, respectively. This results in the formation of five- or six-membered rings. Because the five-membered ring structure resembles the organic molecule furan, derivatives with this structure are termed furanoses. Those with six-membered rings resemble the organic

molecule pyran and are termed pyranoses. Such structures can be depicted by either Fischer or Haworth style diagrams.

The numbering of the carbons in carbohydrates proceeds from the carbonyl carbon, for aldoses, or the carbon nearest the carbonyl, for ketoses.

Cyclic Fischer Projection of αααα-D-Glucose

Haworth Projection of αααα-D-Glucose

The rings can open and re-close, allowing rotation to occur about the carbon bearing the reactive carbonyl yielding two distinct configurations (α and β) of the hemiacetals and hemiketals. The carbon about which this rotation occurs is the anomeric carbon and the two forms are termed anomers. Carbohydrates can

change spontaneously between the α and β configurations-- a process known as mutarotation. When drawn in the Fischer projection, the α configuration places

the hydroxyl attached to the anomeric carbon to the right, towards the ring. When drawn in the Haworth projection, the α configuration places the hydroxyl

downward. The spatial relationships of the atoms of the furanose and pyranose ring

structures are more correctly described by the two conformations identified as the chair form and the boat form. The chair form is the more stable of the two.

Constituents of the ring that project above or below the plane of the ring are axial and those that project parallel to the plane are equatorial. In the chair

conformation, the orientation of the hydroxyl group about the anomeric carbon of α-D-glucose is axial and equatorial in β-D-glucose.

Chair form of αααα-D-Glucose

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Disaccharides

Covalent bonds between the anomeric hydroxyl of a cyclic sugar and the hydroxyl of a second sugar (or another alcohol containing compound) are termed glycosidic bonds, and the resultant molecules are glycosides. The linkage of two monosaccharides to form disaccharides involves a glycosidic bond. Several

physiogically important disaccharides are sucrose, lactose and maltose.

• Sucrose: prevalent in sugar cane and sugar beets, is composed of glucose and fructose through an α-(1,2)β-glycosidic bond.

Sucrose

• Lactose: is found exclusively in the milk of mammals and consists of galactose and glucose in a β-(1,4) glycosidic bond.

Lactose

• Maltose: the major degradation product of starch, is composed of 2 glucose monomers in an α-(1,4) glycosidic bond.

Maltose

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Polysaccharides Most of the carbohydrates found in nature occur in the form of high molecular

weight polymers called polysaccharides. The monomeric building blocks used to generate polysaccharides can be varied; in all cases, however, the

predominant monosaccharide found in polysaccharides is D-glucose. When polysaccharides are composed of a single monosaccharide building block, they are termed homopolysaccharides. Polysaccharides composed of more than

one type of monosaccharide are termed heteropolysaccharides. back to the top

Glycogen

Glycogen is the major form of stored carbohydrate in animals. This crucial molecule is a homopolymer of glucose in α-(1,4) linkage; it is also highly

branched, with α-(1,6) branch linkages occurring every 8-10 residues. Glycogen is a very compact structure that results from the coiling of the polymer chains.

This compactness allows large amounts of carbon energy to be stored in a small

volume, with little effect on cellular osmolarity. back to the top

Starch

Starch is the major form of stored carbohydrate in plant cells. Its structure is identical to glycogen, except for a much lower degree of branching (about every 20-30 residues). Unbranched starch is called amylose; branched starch is called

amylopectin. back to the top



Michael W. King, Ph.D / IU School of Medicine /[email protected]

Last modified: Monday, 18-Aug-2003 16:51:22 EST

• Role of Biological Lipids • Basic Biochemistry of Fatty Acids • Physiologically Relevant Fatty Acids • Basic Structure of Complex Lipids • Triacylglycerides • Phospholipids • Plasmalogens • Sphingolipids • Metabolism of Lipids

o Triacylglycerides o Phospholipids o Sphingolipids o Eicosanoids

• Cholesterol and Bile Acids


Major Roles of Biological of Lipids

Biological molecules that are insoluble in aqueous solutions and soluble in organic solvents are classified as lipids. The lipids of physiological importance for humans have four major functions:

• 1. They serve as structural components of biological membranes. • 2. They provide energy reserves, predominantly in the form of

triacylglycerols. • 3. Both lipids and lipid derivatives serve as vitamins and hormones. • 4. Lipophilic bile acids aid in lipid solubilization.

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Fatty Acids Fatty acids fill two major roles in the body:

• 1. as the components of more complex membrane lipids. • 2. as the major components of stored fat in the form of triacylglycerols.

Fatty acids are long-chain hydrocarbon molecules containing a carboxylic acid moiety at one end. The numbering of carbons in fatty acids begins with the carbon of the carboxylate group. At physiological pH, the carboxyl group is readily ionized, rendering a negative charge onto fatty acids in bodily fluids. Fatty acids that contain no carbon-carbon double bonds are termed saturated fatty acids; those that contain double bonds are unsaturated fatty acids. The numeric designations used for fatty acids come from the number of carbon atoms, followed by the number of sites of unsaturation (eg, palmitic acid is a 16-carbon fatty acid with no unsaturation and is designated by 16:0). The site of unsaturation in a fatty acid is indicated by the symbol ∆∆∆∆ and the number of the first carbon of the double bond (e.g. palmitoleic acid is a 16-carbon fatty acid with one site of unsaturation between carbons 9 and 10, and is designated by 16:1∆9). Saturated fatty acids of less than eight carbon atoms are liquid at physiological temperature, whereas those containing more than ten are solid. The presence of double bonds in fatty acids significantly lowers the melting point relative to a saturated fatty acid. The majority of body fatty acids are acquired in the diet. However, the lipid biosynthetic capacity of the body (fatty acid synthase and other fatty acid modifying enzymes) can supply the body with all the various fatty acid structures needed. Two key exceptions to this are the highly unsaturated fatty acids know as linoleic acid and linolenic acid, containing unsaturation sites beyond carbons 9 and 10. These two fatty acids cannot be synthesized from precursors in the body, and are thus considered the essential fatty acids; essential in the sense that they must be provided in the diet. Since plants are capable of synthesizing linoleic and linolenic acid humans can aquire these fats by consuming a variety of plants or else by eating the meat of animals that have consumed these plant fats. back to the top

Physiologically Relevant Fatty Acids

Numerical

Symbol

Common Name

Structure Comment

s

14:0 Myristic acid CH3(CH2)12COOH

Often found attached to the N-term. of plasma

membrane-associated cytoplasmic

proteins

16:0 Palmitic acid CH3(CH2)14COOH

End product of

mammalian fatty acid synthesis

16:1∆9 Palmitoleic

acid CH3(CH2)5C=C(CH2)7COOH

18:0 Stearic acid CH3(CH2)16COOH

18:1∆9 Oleic acid CH3(CH2)7C=C(CH2)7COOH

18:2∆9,12 Linoleic

acid CH3(CH2)4C=CCH2C=C(CH2)7COOH Essential fatty acid

18:3∆9,12,15 Linolenic

acid CH3CH2C=CCH2C=CCH2C=C(CH2)7CO

OH Essential fatty acid

20:4∆5,8,11,1

4 Arachidoni

c acid CH3(CH2)3(CH2C=C)4(CH2)3COOH

Precursor for

eicosanoid synthesis

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Basic Structure of Triacylglycerides

Triacylglycerides are composed of a glycerol backbone to which 3 fatty acids are esterified.

Basic composition of a triacylglyceride. The glycerol backbone is in blue.</B?< TD>

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Basic Structure of Phospholipids

The basic structure of phospolipids is very similar to that of the triacylglycerides except that C-3 (sn3)of the glycerol backbone is esterified to phosphoric acid. The building block of the phospholipids is phosphatidic acid which results when the X substitution in the basic structure shown in the Figure below is a hydrogen atom. Substitutions include ethanolamine (phosphatidylethanolamine), choline (phosphatidylcholine, also called lecithins), serine (phosphatidylserine), glycerol (phosphatidylglycerol), myo-inositol (phosphatidylinositol, these compounds can have a variety in the numbers of inositol alcohols that are phosphorylated generating polyphosphatidylinositols), and phosphatidylglycerol (diphosphatidylglycerol more commonly known as cardiolipins).

Basic composition of a phospholipid. X can be a number of different substituents.

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Basic Structure of Plasmalogens

Plasmalogens are complex membrane lipids that resemble phospholipids, principally phosphatidylcholine. The major difference is that the fatty acid at C-1 (sn1) of glycerol contains either an O-alkyl or O-alkenyl ether species. A basic O-alkenyl ether species is shown in the Figure below. One of the most potent biological molecules is platelet activating factor (PAF) which is a choline plasmalogen in which the C-2 (sn2) position of glycerol is esterified with an acetyl group insted of a long chain fatty acid.

Top: basic composition of O-alkenyl plasmalogens. Bottom: structure of PAF.

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Basic Structure of Sphingolipids

Sphingolipids are composed of a backbone of sphingosine which is derived itself from glycerol. Sphingosine is N-acetylated by a variety of fatty acids generating a family of molecules referred to as ceramides. Sphingolipids predominate in the myelin sheath of nerve fibers. Sphingomyelin is an abundant sphingolipid

generated by transfer of the phosphocholine moiety of phosphatidylcholine to a ceramide, thus sphingomyelin is a unique form of a phospholipid. The other major class of sphingolipids (besides the sphingomyelins) are the glycosphingolipids generated by substitution of carbohydrates to the sn1 carbon of the glycerol backbone of a ceramide. There are 4 major classes of glycosphingolipids:

� Cerebrosides: contain a single moiety, principally galactose. � Sulfatides: sulfuric acid esters of galactocerebrosides. � Globosides: contain 2 or more sugars. � Gangliosides: similar to globosides except also contain sialic acid.

Top: Sphingosine the atoms in red are derived from glycerol.

Bottom: Basic composition of a ceramide n indicates any fatty acid may be N-acetylated at this position.

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Last modified: Monday, 18-Aug-2003 16:51:25 EST

• Fatty Acid Synthesis • Origin of Acetyl-CoA for Fat Synthesis • Regulation of Fatty Acid Synthesis • Elongation and Desaturation of Fatty Acids • Triacylglyceride Synthesis • Phospholipid Structures • Phospholipid Metabolism • Plasmalogen Synthesis • Sphingolipid Metabolism • Clinical Significances of Sphingolipids • Eicosanoid Metabolism • Properties of the Significant Eicosanoids • Cholesterol and Bile Acid Synthesis • Fatty Acid Oxidation


Fatty Acid Synthesis

One might predict that the pathway for the synthesis of fatty acids would be the reversal of the oxidation pathway. However, this would not allow distinct regulation of the two pathways to occur even given the fact that the pathways are separated within different cellular compartments. The pathway for fatty acid synthesis occurs in the cytoplasm, whereas, oxidation occurs in the mitochondria. The other major difference is the use of nucleotide co-factors. Oxidation of fats involves the reduction of FADH+ and NAD+. Synthesis of fats involves the oxidation of NADPH. However, the essential chemistry of the two processes are reversals of each other. Both oxidation and synthesis of fats utilize an activated two carbon intermediate, acetyl-CoA. However, the acetyl-CoA in fat synthesis exists temporarily bound to the enzyme complex as malonyl-CoA. The synthesis of malonyl-CoA is the first committed step of fatty acid synthesis and the enzyme that catalyzes this reaction, acetyl-CoA carboxylase (ACC), is the major site of regulation of fatty acid synthesis. Like other enzymes that transfer CO2 to substrates, ACC requires a biotin co-factor.

The rate of fatty acid synthesis is controlled by the equilibrium between monomeric ACC and polymeric ACC. The activity of ACC requires polymerization. This conformational change is enhanced by citrate and inhibited by long-chain fatty acids. ACC is also controlled through hormone mediated phosphorylation (see below). The acetyl groups that are the products of fatty acid oxidation are linked to CoASH. As you should recall, CoA contains a phosphopantetheine group coupled to AMP. The carrier of acetyl groups (and elongating acyl groups) during fatty acid synthesis is also a phosphopantetheine prosthetic group, however, it is attached a serine hydroxyl in the synthetic enzyme complex. The carrier portion of the synthetic complex is called acyl carrier protein, ACP. This is somewhat of a misnomer in eukaryotic fatty acid synthesis since the ACP portion of the synthetic complex is simply one of many domains of a single polypeptide. The acetyl-CoA and malonyl-CoA are transferred to ACP by the action of acetyl-CoA transacylase and malonyl-CoA transacylase, respectively. The attachment of these carbon atoms to ACP allows them to enter the fatty acid synthesis cycle. The synthesis of fatty acids from acetyl-CoA and malonyl-CoA is carried out by fatty acid synthase, FAS. The active enzyme is a dimer of identical subunits. All of the reactions of fatty acid synthesis are carried out by the multiple enzymatic activities of FAS. Like fat oxidation, fat synthesis involves 4 enzymatic activities. These are, ββββ-keto-ACP synthase, ββββ-keto-ACP reductase, 3-OH acyl-ACP dehydratase and enoyl-CoA reductase. The two reduction reactions require NADPH oxidation to NADP+. The primary fatty acid synthesized by FAS is palmitate. Palmitate is then released from the enzyme and can then undergo separate elongation and/or unsaturation to yield other fatty acid molecules. back to the top

Origin of Cytoplasmic Acetyl-CoA

Acetyl-CoA is generated in the mitochondria primarily from two sources, the pyruvate dehydrogenase (PDH) reaction and fatty acid oxidation. In order for these acetyl units to be utilized for fatty acid synthesis they must be present in the cytoplasm. The shift from fatty acid oxidation and glycolytic oxidation occurs when the need for energy diminishes. This results in reduced oxidation of acetyl-CoA in the TCA cycle and the oxidative phosphorylation pathway. Under these conditions the mitochondrial acetyl units can be stored as fat for future energy demands. Acetyl-CoA enters the cytoplasm in the form of citrate via the tricarboxylate transport system as diagrammed. In the cytoplasm, citrate is converted to oxaloacetate and acetyl-CoA by the ATP driven ATP-citrate lyase reaction. This

reaction is essentially the reverse of that catalyzed by the TCA enzyme citrate synthase except it requires the energy of ATP hydrolysis to drive it forward. The resultant oxaloacetate is converted to malate by malate dehydrogenase (MDH). The malate produced by this pathway can undergo oxidative decarboxylation by malic enzyme. The co-enzyme for this reaction is NADP+ generating NADPH. The advantage of this series of reactions for converting mitochondrial acetyl-CoA into cytoplasmic acetyl-CoA is that the NADPH produced by the malic enzyme reaction can be a major source of reducing co-factor for the fatty acid synthase activities. back to the top

Regulation of Fatty Acid Metabolism

One must consider the global organismal energy requirements in order to effectively understand how the synthesis and degradation of fats (and also carbohydrates) needs to be exquisitely regulated. The blood is the carrier of triacylglycerols in the form of VLDLs and chylomicrons, fatty acids bound to albumin, amino acids, lactate, ketone bodies and glucose. The pancreas is the primary organ involved in sensing the organisms dietary and energetic states via glucose concentrations in the blood. In response to low blood glucose, glucagon is secreted, whereas, in response to elevated blood glucose insulin is secreted. The regulation of fat metabolism occurs via two distinct mechanisms. One is short term regulation which is regulation effected by events such as substrate availability, allosteric effectors and/or enzyme modification. ACC is the rate limiting (committed) step in fatty acid synthesis. This enzyme is activated by citrate and inhibited by palmitoyl-CoA and other long chain fatty acyl-CoAs. ACC activity also is affected by phosphorylation. The primary phosphorylation of ACC occurs through the action of AMP-activated protein kinase, AMPK (this is not the same as cAMP-dependent protein kinase, PKA). Glucagon stimulated increases in PKA activity result in phosphorylation and inhibition of ACC. Additionally, glucagon activation of PKA leads to phosphorylation and activation of phosphoprotein phosphatase inhibitor-1, PPI-1 which results in a reduced ability to dephosphorylate ACC maintaining the enzyme in a less active state. On the other hand insulin leads to activation of phosphatases, thereby leading to dephosphorylation of ACC that results in increased ACC activity. These forms of regulation are all defined as short term regulation. Control of a given pathways' regulatory enzymes can also occur by alteration of enzyme synthesis and turn-over rates. These changes are long term regulatory effects. Insulin stimulates ACC and FAS synthesis, whereas, starvation leads to decreased synthesis of these enzymes. Adipose tissue lipoprotein lipase levels also are increased by insulin and decreased by starvation. However, in contrast to the effects of insulin and starvation on adipose tissue, their effects on heart lipoprotein lipase are just the inverse. This allows the heart to absorb any available fatty acids in the blood in order to oxidize them for energy production. Starvation also leads to increases in the levels of fatty acid oxidation enzymes in the heart as well as a decrease in FAS and related enzymes of synthesis.

Adipose tissue contains hormone-sensitive lipase, that is activated by PKA-dependent phosphorylation leading to increased fatty acid release to the blood. The activity of hormone-sensitive lipase is also affected positively through the action of AMPK. Both of these effects lead to increased fatty acid oxidation in other tissues such as muscle and liver. In the liver the net result (due to increased acetyl-CoA levels) is the production of ketone bodies. This would occur under conditions where insufficient carbohydrate stores and gluconeogenic precursors were available in liver for increased glucose production. The increased fatty acid availability in response to glucagon or epinephrine is assured of being completely oxidized since both PKA and AMPK also phosphorylate (and as a result inhibits) ACC, thus inhibiting fatty acid synthesis. Insulin, on the other hand, has the opposite effect to glucagon and epi leading to increased glycogen and triacylglyceride synthesis. One of the many effects of insulin is to lower cAMP levels which leads to increased dephosphorylation through the enhanced activity of protein phosphatases such as PP-1. With respect to fatty acid metabolism this yields dephosphorylated and inactive hormone sensitive lipase. Insulin also stimulates certain phosphorylation events. This occurs through activation of several cAMP-independent kinases. Insulin stimulated phosphorylation of ACC activates this enzyme. Regulation of fat metabolism also occurs through malonyl-CoA induced inhibition of carnitine acyltransferase I. This functions to prevent the newly synthesized fatty acids from entering the mitochondria and being oxidized. back to the top

Elongation and Desaturation

The fatty acid product released from FAS is palmitate (via the action of palmitoyl thioesterase) which is a 16:0 fatty acid, i.e. 16 carbons and no sites of unsaturation. Elongation and unsaturation of fatty acids occurs in both the mitochondria and endoplasmic reticulum (microsomal membranes). The predominant site of these processes is in the ER membranes. Elongation involves condensation of acyl-CoA groups with malonyl-CoA. The resultant product is two carbons longer (CO2 is released from malonyl-CoA as in the FAS reaction) which undergoes reduction, dehydration and reduction yielding a saturated fatty acid. The reduction reactions of elongation require NADPH as co-factor just as for the similar reactions catalyzed by FAS. Mitochondrial elongation involves acetyl-CoA units and is a reversal of oxidation except that the final reduction utilizes NADPH instead of FADH2 as co-factor. Desaturation occurs in the ER membranes as well and in mammalian cells involves 4 broad specificity fatty acyl-CoA desaturases (non-heme iron containing enzymes). These enzymes introduce unsaturation at C4, C5, C6 or C9. The electrons transferred from the oxidized fatty acids during desaturation are transferred from the desaturases to cytochrome b5 and then NADH-cytochrome b5 reductase. These electrons are un-coupled from mitochondrial oxidative-phosphorylation and, therefore, do not yield ATP. Since these enzymes cannot introduce sites of unsaturation beyond C9 they cannot synthesize either linoleate (18:2∆9, 12) or linolenate (18:3∆9, 12, 15). These

fatty acids must be acquired from the diet and are, therefore, referred to as essential fatty acids. Linoleic is especially important in that it required for the synthesis of arachidonic acid. As we shall encounter later, arachindonate is a precursor for the eicosanoids (the prostaglandins and thromboxanes). It is this role of fatty acids in eicosanoid synthesis that leads to poor growth, wound healing and dermatitis in persons on fat free diets. Also, linoleic acid is a constituent of epidermal cell sphingolipids that function as the skins water permeability barrier. back to the top

Synthesis of Triglycerides

Fatty acids are stored for future use as triacylglycerols in all cells, but primarily in adipocytes of adipose tissue. Triacylglycerols constitute molecules of glycerol to which three fatty acids have been esterified. The fatty acids present in triacylglycerols are predominantly saturated. The major building block for the synthesis of triacylglycerols, in tissues other than adipose tissue, is glycerol. Adipocytes lack glycerol kinase, therefore, dihydroxyacetone phosphate (DHAP), produced during glycolysis, is the precursor for triacylglycerol synthesis in adipose tissue. This means that adipoctes must have glucose to oxidize in order to store fatty acids in the form of triacylglycerols. DHAP can also serve as a backbone precursor for triacylglycerol synthesis in tissues other than adipose, but does so to a much lesser extent than glycerol.

The glycerol backbone of triacylglycerols is activated by phosphorylation at the C-3 position by glycerol kinase. The utilization of DHAP for the backbone is carried out through the action of glycerol-3-phosphate dehydrogenase, a reaction that requires NADH (the same reaction as that used in the glycerol-phosphate shuttle). The fatty acids incorporated into triacylglycerols are activated to acyl-CoAs through the action of acyl-CoA synthetases. Two molecules of acyl-CoA are esterified to glycerol-3-phosphate to yield 1,2-diacylglycerol phosphate (commonly identified as phosphatidic acid). The phosphate is then removed, by phosphatidic acid phosphatase, to yield 1,2-diacylglycerol, the substrate for addition of the third fatty acid. Intestinal monoacylglycerols, derived from the hydrolysis of dietary fats, can also serve as substrates for the synthesis of 1,2-diacylglycerols. back to the top

Phospholipid Structures

Phospholipids are synthesized by esterification of an alcohol to the phosphate of phosphatidic acid (1,2-diacylglycerol 3-phosphate). Most phospholipids have a saturated fatty acid on C-1 and an unsaturated fatty acid on C-2 of the glycerol backbone. The most commonly added alcohols (serine, ethanolamine and choline) also contain nitrogen that may be positively charged, whereas, glycerol and inositol do not. The major classifications of phospholipids are:

Phosphatidylcholine (PC)

Phosphatidylethanolamine (PE)

Phosphatidylserine (PS)

Phosphatidylinositol (PI)

Phosphatidylglycerol (PG)

Diphosphatidylglycerol (DPG)

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Phospholipid Synthesis

Phospholipids can be synthesized by two mechanisms. One utilizes a CDP-activated polar head group for attachment to the phosphate of phosphatidic acid. The other utilizes CDP-activated 1,2-diacylglycerol and an inactivated polar head group. PC:This class of phospholipids is also called the lecithins. At physiological pH, phosphatidylcholines are neutral zwitterions. They contain primarily palmitic or stearic acid at carbon 1 and primarily oleic, linoleic or linolenic acid at carbon 2. The lecithin dipalmitoyllecithin is a component of lung or pulmonary surfactant. It contains palmitate at both carbon 1 and 2 of glycerol and is the major (80%) phospholipid found in the extracellular lipid layer lining the pulmonary alveoli. Choline is activated first by phosphorylation and then by coupling to CDP prior to attachment to phosphatidic acid. PC is also synthesized by the addition of choline to CDP-activated 1,2-diacylglycerol. A third pathway to PC synthesis, involves the conversion of either PS or PE to PC. The conversion of PS to PC

first requires decarboxylation of PS to yield PE; this then undergoes a series of three methylation reactions utilizing S-adenosylmethionine (SAM) as methyl group donor. PE:These molecules are neutral zwitterions at physiological pH. They contain primarily palmitic or stearic acid on carbon 1 and a long chain unsaturated fatty acid (e.g. 18:2, 20:4 and 22:6) on carbon 2. Synthesis of PE can occur by two pathways. The first requires that ethanolamine be activated by phosphorylation and then by coupling to CDP. The ethanolamine is then transferred from CDP-ethanolamine to phosphatidic acid to yield PE. The second involves the decarboxylation of PS. PS:Phosphatidylserines will carry a net charge of -1 at physiological pH and are composed of fatty acids similar to the phosphatidylethanolamines. The pathway for PS synthesis involves an exchange reaction of serine for ethanolamine in PE. This exchange occurs when PE is in the lipid bilayer of the a membrane. As indicated above, PS can serve as a source of PE through a decarboxylation reaction. PI:These molecules contain almost exclusively stearic acid at carbon 1 and arachidonic acid at carbon 2. Phosphatidylinositols composed exclusively of non-phosphorylated inositol exhibit a net charge of -1 at physiological pH. These molecules exist in membranes with various levels of phosphate esterified to the hydroxyls of the inositol. Molecules with phosphorylated inositol are termed polyphosphoinositides. The polyphosphoinositides are important intracellular transducers of signals emanating from the plasma membrane. The synthesis of PI involves CDP-activated 1,2-diacylglycerol condensation with myo-inositol. PI subsequently undergoes a series of phosphorylations of the hydroxyls of inositol leading to the production of polyphosphoinositides. One polyphosphoinositide (phosphatidylinositol 4,5-bisphosphate, PIP2) is a critically important membrane phospholipid involved in the transmission of signals for cell growth and differentiation from outside the cell to inside. PG:Phosphatidylglycerols exhibit a net charge of -1 at physiological pH. These molecules are found in high concentration in mitochondrial membranes and as components of pulmonary surfactant. Phosphatidylglycerol also is a precursor for the synthesis of cardiolipin. PG is synthesized from CDP-diacylglycerol and glycerol-3-phosphate. The vital role of PG is to serve as the precursor for the synthesis of diphosphatidylglycerols (DPGs). DPG:These molecules are very acidic, exhibiting a net charge of -2 at physiological pH. They are found primarily in the inner mitochondrial membrane and also as components of pulmonary surfactant. One important class of diphosphatidylglycerols is the cardiolipins. These molecules are synthesized by the condensation of CDP-diacylglycerol with PG. The fatty acid distribution at the C-1 and C-2 positions of glycerol within phospholipids is continually in flux, owing to phospholipid degradation and the continuous phospholipid remodeling that occurs while these molecules are in membranes. Phospholipid degradation results from the action of

phospholipases. There are various phospholipases that exhibit substrate specificities for different positions in phospholipids. In many cases the acyl group which was initially transferred to glycerol, by the action of the acyl transferases, is not the same acyl group present in the phospholipid when it resides within a membrane. The remodeling of acyl groups in phospholipids is the result of the action of phospholipase A1 and phospholipase A2.

Sites of action of the phospholipases A1, A2, C and D.

The products of these phospholipases are called lysophospholipids and can be substrates for acyl transferases utilizing different acyl-CoA groups. Lysophospholipids can also accept acyl groups from other phospholipids in an exchange reaction catalyzed by lysolecithin:lecithin acyltransferase (LLAT). Phospholipase A2 is also an important enzyme, whose activity is responsible for the release of arachidonic acid from the C-2 position of membrane phospholipids. The released arachidonate is then a substrate for the synthesis of the prostaglandins and leukotrienes. back to the top

Plasmalogens

Plasmalogens are glycerol ether phospholipids. They are of two types, alkyl ether and alkenyl ether. Dihydroxyacetone phosphate serves as the glycerol precursor for the synthesis of glycerol ether phospholipids. Three major classes of plasmalogens have been identified: choline, ethanolamine and serine plasmalogens. Ethanolamine plasmalogen is prevalent in myelin. Choline plasmalogen is abundant in cardiac tissue. One particular choline plasmalogen

(1-alkyl, 2-acetyl phosphatidylcholine) has been identified as an extremely powerful biological mediator, capable of inducing cellular responses at concentrations as low as 10-11 M. This molecule is called platelet activating factor, PAF.

Platelet activating factor

PAF functions as a mediator of hypersensitivity, acute inflammatory reactions and anaphylactic shock. PAF is synthesized in response to the formation of antigen-IgE complexes on the surfaces of basophils, neutrophils, eosinophils, macrophages and monocytes. The synthesis and release of PAF from cells leads to platelet aggregation and the release of serotonin from platelets. PAF also produces responses in liver, heart, smooth muscle, and uterine and lung tissues. back to the top

Metabolism of the Sphingolipids

The sphingolipids, like the phospholipids, are composed of a polar head group and two nonpolar tails. The core of sphingolipids is the long-chain amino alcohol, sphingosine. Amino acylation, with a long chain fatty acid, at carbon 2 of sphingosine yields a ceramide.

Top: Sphingosine Bottom: Ceramide

The sphingolipids include the sphingomyelins and glycosphingolipids (the cerebrosides, sulfatides, globosides and gangliosides). Sphingomyelins are the only sphingolipid that are phospholipids. Sphingolipids are a component of all membranes but are particularly abundant in the myelin sheath. Sphingomyelins are sphingolipids that are also phospholipids. Sphingomyelins are important structural lipid components of nerve cell membranes. The predominant sphingomyelins contain palmitic or stearic acid N-acylated at carbon 2 of sphingosine. The sphingomyelins are synthesized by the transfer of phosphorylcholine from phosphatidylcholine to a ceramide in a reaction catalyzed by sphingomyelin synthase.

A sphingomyelin

Defects in the enzyme acid sphingomyelinase result in the lysosomal storage disease known as Niemann-Pick disease. There are at least 4 related disorders identified as Niemann-Pick disease Type A and B (both of which result from defects in acid sphingomyelinase), Type C1 and a related C2 and Type D. Types C1, C2 and D do not result from defects in acid sphingomyelinase. More information on Niemann-Pick sub-type C1 is presented below in the section on Clinical Significances of Sphinoglipids.

Glycosphingolipids, or glycolipids, are composed of a ceramide backbone with a wide variety of carbohydrate groups (mono- or oligosaccharides) attached to carbon 1 of sphingosine. The four principal classes of glycosphingolipids are the cerebrosides, sulfatides, globosides and gangliosides. Cerebrosides have a single sugar group linked to ceramide. The most common of these is galactose (galactocerebrosides), with a minor level of glucose (glucocerebrosides). Galactocerebrosides are found predominantly in neuronal cell membranes. By contrast glucocerebrosides are not normally found in membranes, especially neuronal membranes; instead, they represent intermediates in the synthesis or degradation of more complex glycosphingolipids. Galactocerebrosides are synthesized from ceramide and UDP-galactose. Excess accumulation of glucocerebrosides is observed in Gaucher's disease.

A Galactocerebroside

Sulfatides: The sulfuric acid esters of galactocerebrosides are the sulfatides. Sulfatides are synthesized from galactocerebrosides and activated sulfate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). Excess accumulation of sulfatides is observed in sulfatide lipidosis (metachromatic leukodystrophy). Globosides: Globosides represent cerebrosides that contain additional carbohydrates, predominantly galactose, glucose or GalNAc. Lactosyl ceramide is a globoside found in erythrocyte plasma membranes. Globotriaosylceramide (also called ceramide trihexoside) contains glucose and two moles of galactose and accumulates, primarily in the kidneys, of patients suffering from Fabry's disease. Gangliosides: Gangliosides are very similar to globosides except that they also contain NANA in varying amounts. The specific names for gangliosides are a key to their structure. The letter G refers to ganglioside, and the subscripts M, D, T and Q indicate that the molecule contains mono-, di-, tri and quatra(tetra)-sialic acid. The numerical subscripts 1, 2 and 3 refer to the carbohydrate sequence that is attached to ceramide; 1 stands for GalGalNAcGalGlc-ceramide, 2 for GalNAcGalGlc-ceramide and 3 for GalGlc-ceramide. Deficiencies in lysosomal enzymes, which normally are responsible for the degradation of the carbohydrate portions of various gangliosides, underlie the symptoms observed in rare autosomally inherited diseases termed lipid storage

diseases, many of which are listed below. back to the top

Clinical Significances of Sphingolipids

One of the most clinically important classes of sphingolipids are those that confer antigenic determinants on the surfaces of cells, particularly the erythrocytes. The ABO blood group antigens are the carbohydrate moieties of glycolipids on the surface of cells as well as the carbohydrate portion of serum glycoproteins. When present on the surface of cells the ABO carbohydrates are linked to sphingolipid and are therefore of the glycosphingolipid class. When the ABO carbohydrates are associated with protein in the form of glycoproteins they are found in the serum and are referred to as the secreted forms. Some individuals produce the glycoprotein forms of the ABO antigens while others do not. This property distinguishes secretors from non-secretors, a property that has forensic importance such as in cases of rape.

Structure of the ABO blood group carbohydrates, with sialylated Lewis antigen also shown.

Image copyright M.W. King 2003

R represents the linkage to protein in the secreted forms, sphingolipid in the cell-surface bound form.

open square = GlcNAc, open diamond = galactose, filled square = fucose, filled diamond = GalNAc, filled diamond = sialic acid (NANA)

A significant cause of death in premature infants and, on occasion, in full term infants is respiratory distress syndrome (RDS) or hyaline membrane disease. This condition is caused by an insufficient amount of pulmonary surfactant. Under normal conditions the surfactant is synthesized by type II endothelial cells and is secreted into the alveolar spaces to prevent atelectasis following expiration during breathing. Surfactant is comprised primarily of dipalmitoyllecithin; additional lipid components include phosphatidylglycerol and phosphatidylinositol along with proteins of 18 and 36 kDa (termed surfactant proteins). During the third trimester the fetal lung synthesizes primarily sphingomyelin, and type II endothelial cells convert the majority of their stored glycogen to fatty acids and then to dipalmitoyllecithin. Fetal lung maturity can be determined by measuring the ratio of lecithin to sphingomyelin (L/S ratio) in the amniotic fluid. An L/S ratio less than 2.0 indicates a potential risk of RDS. The risk is nearly 75-80% when the L/S ratio is 1.5. The carbohydrate portion of the ganglioside, GM1, present on the surface of intestinal epithelial cells, is the site of attachment of cholera toxin, the protein secreted by Vibrio cholerae. These are just a few examples of how sphingolipids and glycosphingolipids are involved in various recognition functions at the surface of cells. As with the complex glycoproteins, an understanding of all of the functions of the glycolipids is far from complete.

Disorders Associated with Abnormal Sphingolipid Metabolism

Disorder Enzyme

Deficiency Accumulating

Substance Symptoms

Tay-Sachs disease

see below table HEXA GM2 ganglioside

rapidly progressing mental retardation, blindness, early mortality

Sandhoff-Jatzkewitz

disease see below table

HEXB Globoside, GM2 ganglioside

same symptoms as Tay-Sachs, progresses more rapidly

Tay-Sachs AB variant

see below table

GM2 activator (GM2A) GM2 ganglioside same symptoms

as Tay-Sachs

Gaucher's disease

Glucocerebrosidase Glucocerebroside

hepatosplenomegaly, mental retardation in infantile form, long bone degeneration

Fabry's disease α-Galactosidase A

Globotriaosylceramide; also called ceramide

trihexoside (CTH)

kidney failure, skin rashes

Niemann-Pick disease, more

info below Types A and B

Type C1 Type C2 Type D

Sphingomyelinase see info below see info below

Sphingomyelin LDL-derived cholesterol

LDL-derived cholesterol

all types lead to mental retardation, hepatosplenomegaly, early fatality potential

Krabbe's disease; globoid

leukodystrophy

Galactocerebrosidase Galactocerebroside

mental retardation, myelin deficiency

GM1 gangliosidosis

GM1 ganglioside:β -galactosidase

GM1 ganglioside

mental retardation, skeletal abnormalities, hepatomegaly

Sulfatide lipodosis;

metachromatic leukodystrophy

Arylsulfatase A Sulfatide

mental retardation, metachromasia of nerves

Fucosidosis α-L-Fucosidase Pentahexosylfucoglyc

olipid

cerebral degeneration, thickened skin, muscle spasticity

Farber's lipogranulomat

osis Acid ceramidase Ceramide

hepatosplenomegaly, painful swollen joints

The GM2 gangliosidoses include Tay-Sachs disease, the Sandhoff diseases and the GM2 activator deficiencies. GM2 ganglioside degradation requires the enzyme ββββ-hexosaminidase and the GM2 activator protein (GM2A). Hexosaminidase is a dimer composed of 2 subunits, either α and/or β. The HexS protein is αα, HexA is αβ and HexB is ββ. It is the α-subunit that carries out the catalysis of GM2 gangliosides. The activator first binds to GM2 gangliosides followed by hexosaminidase and then digestion occurs. Based upon genetic linkage analyses as well as enzyme studies and the characterization of accumulating lysosomal substances, Niemann Pick disease should be divided into type I and type II; type I has 2 subtypes, A and B (NPA and NPB), which show deficiency of acid sphingomyelinase. Niemann Pick disease type II likewise has 2 subtypes, type C1 and C2 (NPC) and type D (NPD). It is obviously confusing to use the abbreviation NPD for Niemann Pick disease in some cases and for subtype D of Niemann Pick disease in other cases. Recent studies (Science vol. 277 pp. 228-231 and 232-235: July 11, 1997) identified the gene for NPC1. This gene contains regions of homology to mediators of cholesterol homeostasis suggesting why LDL-cholesterol accumulates in lysosomes of afflicted individuals. The encoded protein product of NPC1 gene is 1278 amino acids long. Within the protein are regions of homology to the transmembrane domain of the morphogen receptor patched (of Drosophila melanogaster), the sterol-sensing domain of SREBP (sterol regulated element binding protein) cleavage-activating protein, SCAP and HMG-CoA reductase. back to the top

Metabolism of the Eicosanoids

The eicosanoids consist of the prostaglandins (PGs), thromboxanes (TXs) and leukotrienes (LTs). The PGs and TXs are collectively identified as prostanoids. Prostaglandins were originally shown to be synthesized in the prostate gland, thromboxanes from platelets (thrombocytes) and leukotrienes from leukocytes, hence the derivation of their names.

Structures of Representive Clinically Relevant Eicosanoids

PGE2

TXA2

LTA4

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The eicosanoids produce a wide range of biological effects on inflammatory responses (predominantly those of the joints, skin and eyes), on the intensity and duration of pain and fever, and on reproductive function (including the induction

of labor). They also play important roles in inhibiting gastric acid secretion, regulating blood pressure through vasodilation or constriction, and inhibiting or

activating platelet aggregation and thrombosis. The principal eicosanoids of biological significance to humans are a group of

molecules derived from the C20 fatty acid, arachidonic acid. Minor eicosanoids are derived from eicosopentaenoic acid which is itself derived from α-linolenic acid obtained in the diet. The major source of arachidonic acid is through its

release from cellular stores. Within the cell, it resides predominantly at the C-2 position of membrane phospholipids and is released from there upon the

activation of phospholipase A2 (see diagram above). The immediate dietary precursor of arachidonate is linoleic acid. Linoleic acid is converted to

arachidonic acid through the steps outlined in the figure below. Linoleic acid (arachidonate precursor) and α-linolenic acid (eicosapentaenoate precursor) are

essential fatty acids, therefore, their absence from the diet would seriously threaten the body's ability to synthesize eicosanoids.

Pathway from linoleic acid to arachidonic acid. Numbers in parentheses refer to the fatty acid length and the number and positions of unsaturations.

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All mammalian cells except erythrocytes synthesize eicosanoids. These molecules are extremely potent, able to cause profound physiological effects at

very dilute concentrations. All eicosanoids function locally at the site of synthesis, through receptor-mediated G-protein linked signaling pathways leading to an

increase in cAMP levels. Two main pathways are involved in the biosynthesis of eicosanoids. The

prostaglandins and thromboxanes are synthesized by the cyclic pathway, the leukotrienes by the linear pathway.

Synthesis of the clinically relevant prostaglandins and thromboxanes from arachidonic acid. Numerous stimuli (e.g. epinephrine, thrombin and bradykinin) activate phospholipase A2 which hydrolyzes arachidonic acid from membrane phospholipids. The prostaglandins are identified as PG and the thromboxanes as TX. Prostaglandin PGI2 is also known as prostacyclin. The subscript 2 in each molecule refers to the number of -C=C- present.

Synthesis of the clinically relevant leukotrienes from arachidonic acid. Numerous stimuli (e.g. epinephrine, thrombin and bradykinin) activate phospholipase A2 which hydrolyzes arachidonic acid from membrane phospholipids. The leukotrienes are identified as LT. The leukotrienes, LTC4, LTD4, LTE4 and LTF4 are known as the peptidoleukotrienes because of the presence of amino acids. The peptidoleukotrienes, LTC4, LTD4 and LTE4 are components of slow-reacting substance of anaphylaxis The subscript 4 in each molecule refers to the number of -C=C- present.

The cyclic pathway is initiated through the action of prostaglandin G/H synthase, PGS (also called prostaglandin endoperoxide synthetase). This enzyme possesses two activities, cyclooxygenase (COX) and peroxidase.

There are 2 forms of the COX activity. COX-1 (PGS-1) is expressed constitutively in gastric mucosa, kidney, platelets, and vascular endothelial cells. COX-2 (PGS-2) is inducible and is expressed in macrophages and monocytes in response to

inflammation. The primary trigger for COX-2 induction in monocytes and macrophages is platelet-activating factor, PAF and interleukin-1, IL-1. Both

COX-1 and COX-2 catalyze the 2-step conversion of arachidonic acid to PGG2 and then to PGH2.

The linear pathway is initiated through the action of lipoxygenases. It is the enzyme, 5-lipoxygenase that gives rise to the leukotrienes.

A widely used class of drugs, the non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen, indomethacin, naproxen, phenylbutazone and aspirin, all act upon the cyclooxygenase activity, inhibiting both COX-1 and COX-2. Because

inhibition of COX-1 activity in the gut is associated with NSAID-induced ulcerations, pharmaceutical companies have developed drugs targeted

exclusively against the inducible COX-2 activity (e.g. celecoxib and rofecoxib). Another class, the corticosteroidal drugs, act to inhibit phospholipase A2,

thereby inhibiting the release of arachidonate from membrane phospholipids and the subsequent synthesis of eicosinoids.

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Properties of Significant Eicosanoids

Eicosanoid Major site(s) of

synthesis Major biological activities

PGD2 mast cells

inhibits platelet and leukocyte aggregation, decreases T-cell

proliferation and lymphocyte migration and secretion of IL-1α and IL-2; induces

vasodilation and production of cAMP

PGE2 kidney, spleen, heart

increases vasodilation and cAMP production, enhancement of the effects

of bradykinin and histamine, induction of uterine contractions and of platelet aggregation, maintaining the open

passageway of the fetal ductus arteriosus; decreases T-cell proliferation and lymphocyte migration and secretion

of IL-1α and IL-2

PGF2α kidney, spleen,

heart

increases vasoconstriction, bronchoconstriction and smooth muscle

contraction

PGH2 precursor to thromboxanes A2 and B2, induction of platelet aggregation and

vasoconstriction

PGI2 heart, vascular endothelial cells

inhibits platelet and leukocyte aggregation, decreases T-cell

proliferation and lymphocyte migration and secretion of IL-1α and IL-2; induces

vasodilation and production of cAMP

TXA2 platelets induces platelet aggregation, vasoconstriction, lymphocyte

proliferation and bronchoconstriction

TXB2 platelets induces vasoconstriction

LTB4

monocytes, basophils, neutrophils, eosinophils, mast cells, epithelial cells

induces leukocyte chemotaxis and aggregation, vascular permeability, T-

cell proliferation and secretion of INF-γ, IL-1 and IL-2

LTC4

monocytes and alveolar macrophages, basophils, eosinophils, mast cells, epithelial cells

component of SRS-A, microvascular vasoconstrictor, vascular permeability

and bronchoconstriction and secretion of INF-γ

LTD4

monocytes and alveolar macrophages, eosinophils, mast cells, epithelial cells

predominant component of SRS-A, microvascular vasoconstrictor, vascular

permeability and bronchoconstriction and secretion of INF-γ

LTE4 mast cells and basophils

component of SRS-A, microvascular vasoconstrictor and bronchoconstriction

**SRS-A = slow-reactive substance of anaphylaxis back to the top



Last modified: Tuesday, 04-Nov-2003 11:34:38 EST

• Introduction to Nucleic Acids • Nucleic Acid Structure and Nomenclature • Adenosine Derivatives • Guanosine Derivatives • Nucleotide Analogs • Polynucleotides • The Structure of DNA

o Thermal Properties of the Double Helix • Analytical Tools for DNA Study

o Chromatography o Electrophoresis


Introduction

As a class, the nucleotides may be considered one of the most important metabolites of the cell. Nucleotides are found primarily as the monomeric units comprising the major nucleic acids of the cell, RNA and DNA. However, they also are required for numerous other important functions within the cell. These functions include:

• 1. serving as energy stores for future use in phosphate transfer reactions. These reactions are predominantly carried out by ATP.

• 2. forming a portion of several important coenzymes such as NAD+, NADP+, FAD and coenzyme A.

• 3. serving as mediators of numerous important cellular processes such as second messengers in signal transduction events. The predominant second messenger is cyclic-AMP (cAMP), a cyclic derivative of AMP formed from ATP.

• 4. controlling numerous enzymatic reactions through allosteric effects on enzyme activity.

• 5. serving as activated intermediates in numerous biosynthetic reactions. These activated intermediates include S-adenosylmethionine (S-AdoMet) involved in methyl transfer reactions as well as the many sugar coupled nucleotides involved in glycogen and glycoprotein synthesis.

back to the top

Nucleoside and Nucleotide Structure and Nomenclature

The nucleotides found in cells are derivatives of the heterocyclic highly basic, compounds, purine and pyrimidine.

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Purine Pyrimidine

It is the chemical basicity of the nucleotides that has given them the common

term "bases" as they are associated with nucleotides present in DNA and RNA. There are five major bases found in cells. The derivatives of purine are called adenine and guanine, and the derivatives of pyrimidine are called thymine,

cytosine and uracil. The common abbreviations used for these five bases are, A, G, T, C and U.

Base Formula Base (X=H)

Nucleoside X=ribose or deoxyribose

Nucleotide X=ribose

phosphate

Cytosine, C Cytidine, A Cytidine

monophosphate CMP

Uracil, U Uridine, U Uridine

monophosphate UMP

Thymine, T Thymidine, T Thymidine

monophosphate TMP

Adenine, A Adenosine, A Adenosine

monophosphate AMP

Guanine, G Guanosine, A Guanosine

monophosphate GMP

The purine and pyrimidine bases in cells are linked to carbohydrate and in this form are termed, nucleosides. The nucleosides are coupled to D-ribose or 2'-deoxy-D-ribose through a β-N-glycosidic bond between the anomeric carbon of

the ribose and the N9 of a purine or N1 of a pyrimidine. The base can exist in 2 distinct orientations about the N-glycosidic bond. These

conformations are identified as, syn and anti. It is the anti conformation that predominates in naturally occurring nucleotides.

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syn-Adenosine anti-Adenosine

Nucleosides are found in the cell primarily in their phosphorylated form. These

are termed nucleotides. The most common site of phosphorylation of nucleotides found in cells is the hydroxyl group attached to the 5'-carbon of the

ribose The carbon atoms of the ribose present in nucleotides are designated with a prime (') mark to distinguish them from the backbone numbering in the bases.

Nucleotides can exist in the mono-, di-, or tri-phosphorylated forms. Nucleotides are given distinct abbreviations to allow easy identification of their

structure and state of phosphorylation. The monophosphorylated form of adenosine (adenosine-5'-monophosphate) is written as, AMP. The di- and tri-phosphorylated forms are written as, ADP and ATP, respectively. The use of

these abbreviations assumes that the nucleotide is in the 5'-phosphorylated form. The di- and tri-phosphates of nucleotides are linked by acid anhydride bonds.

Acid anhydride bonds have a high ∆G0' for hydrolysis imparting upon them a high potential to transfer the phosphates to other molecules. It is this property of the

nucleotides that results in their involvement in group transfer reactions in the cell. The nucleotides found in DNA are unique from those of RNA in that the ribose exists in the 2'-deoxy form and the abbreviations of the nucleotides contain a d

designation. The monophosphorylated form of adenosine found in DNA (deoxyadenosine-5'-monophosphate) is written as dAMP.

The nucleotide uridine is never found in DNA and thymine is almost exclusively found in DNA. Thymine is found in tRNAs but not rRNAs nor mRNAs. There are several less common bases found in DNA and RNA. The primary modified base

in DNA is 5-methylcytosine. A variety of modified bases appear in the tRNAs. Many modified nucleotides are encountered outside of the context of DNA and

RNA that serve important biological functions. back to the top

Adenosine Derivatives

The most common adenosine derivative is the cyclic form, 3'-5'-cyclic adenosine monophosphate, cAMP. This compound is a very powerful second

messenger involved in passing signal transduction events from the cell surface to internal proteins, e.g. cAMP-dependent protein kinase (PKA). PKA

phosphorylates a number of proteins, thereby, affecting their activity either positively or negatively. Cyclic-AMP is also involved in the regulation of ion

channels by direct interaction with the channel proteins, e.g. in the activation of odorant receptors by odorant molecules.

Formation of cAMP occurs in response to activation of receptor coupled adenylate cyclase. These receptors can be of any type, e.g. hormone receptors

or odorant receptors. S-adenosylmethionine is a form of activated methionine which serves as a methyl donor in methylation reactions and as a source of propylamine in the

synthesis of polyamines. back to the top

Guanosine Derivatives

A cyclic form of GMP (cGMP) also is found in cells involved as a second messenger molecule. In many cases its' role is to antagonize the effects of cAMP. Formation of cGMP occurs in response to receptor mediated signals similar to those for activation of adenylate cyclase. However, in this case it is

guanylate cyclase that is coupled to the receptor. The most important cGMP coupled signal transduction cascade is that

photoreception. However, in this case activation of rhodopsin (in the rods) or other opsins (in the cones) by the absorption of a photon of light (through 11-cis-

retinal covalently associated with rhodopsin and opsins) activates transducin which in turn activates a cGMP specific phosphodiesterase that hydrolyzes

cGMP to GMP. This lowers the effective concentration of cGMP bound to gated ion channels resulting in their closure and a concomitant hyperpolarization of the

cell. back to the top

Synthetic Nucleotide Analogs

Many nucleotide analogues are chemically synthesized and used for their therapeutic potential. The nucleotide analogues can be utilized to inhibit specific enzymatic activities. A large family of analogues are used as anti-tumor agents,

for instance, because they interfere with the synthesis of DNA and thereby preferentially kill rapidly dividing cells such as tumor cells. Some of the nucleotide

analogues commonly used in chemotherapy are 6-mercaptopurine, 5-fluorouracil, 5-iodo-2'-deoxyuridine and 6-thioguanine. Each of these compounds disrupts the normal replication process by interfering with the formation of correct

Watson-Crick base-pairing. Nucleotide analogs also have been targeted for use as antiviral agents. Several

analogs are used to interfere with the replication of HIV, such as AZT (azidothymidine) and ddI (dideoxyinosine).

Several purine analogs are used to treat gout. The most common is allopurinol, which resembles hypoxanthine. Allopurinol inhibits the activity of xanthine

oxidase, an enzyme involved in de novo purine biosynthesis. Additionally, several nucleotide analogues are used after organ transplantation in order to

suppress the immune system and reduce the likelihood of transplant rejection by the host.

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Polynucleotides Polynucleotides are formed by the condensation of two or more nucleotides. The condensation most commonly occurs between the alcohol of a 5'-phosphate of

one nucleotide and the 3'-hydroxyl of a second, with the elimination of H2O, forming a phosphodiester bond. The formation of phosphodiester bonds in

DNA and RNA exhibits directionality. The primary structure of DNA and RNA (the linear arrangement of the nucleotides) proceeds in the 5' ----> 3' direction. The common representation of the primary structure of DNA or RNA molecules is to write the nucleotide sequences from left to right synonymous with the 5' -----> 3'

direction as shown: 5'-pGpApTpC-3'

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Structure of DNA

Utilizing X-ray diffraction data, obtained from crystals of DNA, James Watson and Francis Crick proposed a model for the structure of DNA. This model

(subsequently verified by additional data) predicted that DNA would exist as a helix of two complementary antiparallel strands, wound around each other in a

rightward direction and stabilized by H-bonding between bases in adjacent strands. In the Watson-Crick model, the bases are in the interior of the helix

aligned at a nearly 90 degree angle relative to the axis of the helix. Purine bases form hydrogen bonds with pyrimidines, in the crucial phenomenon of base

pairing. Experimental determination has shown that, in any given molecule of DNA, the concentration of adenine (A) is equal to thymine (T) and the

concentration of cytidine (C) is equal to guanine (G). This means that A will only base-pair with T, and C with G. According to this pattern, known as Watson-Crick base-pairing, the base-pairs composed of G and C contain three H-

bonds, whereas those of A and T contain two H-bonds. This makes G-C base-pairs more stable than A-T base-pairs.

A-T Base Pair G-C Base Pair

The antiparallel nature of the helix stems from the orientation of the individual strands. From any fixed position in the helix, one strand is oriented in the 5' ---> 3' direction and the other in the 3' ---> 5' direction. On its exterior surface, the

double helix of DNA contains two deep grooves between the ribose-phosphate chains. These two grooves are of unequal size and termed the major and minor grooves. The difference in their size is due to the asymmetry of the deoxyribose

rings and the structurally distinct nature of the upper surface of a base-pair relative to the bottom surface.

The double helix of DNA has been shown to exist in several different forms, depending upon sequence content and ionic conditions of crystal preparation.

The B-form of DNA prevails under physiological conditions of low ionic strength and a high degree of hydration. Regions of the helix that are rich in pCpG

dinucleotides can exist in a novel left-handed helical conformation termed Z-DNA. This conformation results from a 180 degree change in the orientation of

the bases relative to that of the more common A- and B-DNA.

Structure of B-DNA Structure of Z-DNA

Parameters of Major DNA Helices

Parameters A Form B Form Z-Form

Direction of helical rotation Right Right Left

Residues per turn of helix 11 10 12 base pairs

Rotation of helix per residue (in degrees) 33 36 -30

Base tilt relative to helix axis (in degrees) 20 6 7

Major groove narrow and deep wide and deep Flat

Minor groove wide and shallow

narrow and deep narrow and deep

Orientation of N- Anti Anti Anti for Py, Syn for Pu

glycosidic Bond

Comments most prevalent within cells

occurs in stretches of alternating purine-

pyrimidine base pairs

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Thermal Properties of DNA

As cells divide it is a necessity that the DNA be copied (replicated), in such a way that each daughter cell acquires the same amount of genetic material. In order

for this process to proceed the two strands of the helix must first be separated, in a process termed denaturation. This process can also be carried out in vitro. If a solution of DNA is subjected to high temperature, the H-bonds between bases become unstable and the strands of the helix separate in a process of thermal

denaturation. The base composition of DNA varies widely from molecule to molecule and even

within different regions of the same molecule. Regions of the duplex that have predominantly A-T base-pairs will be less thermally stable than those rich in G-C base-pairs. In the process of thermal denaturation, a point is reached at which 50% of the DNA molecule exists as single strands. This point is the melting temperature (TM), and is characteristic of the base composition of that DNA

molecule. The TM depends upon several factors in addition to the base composition. These include the chemical nature of the solvent and the identities

and concentrations of ions in the solution. When thermally melted DNA is cooled, the complementary strands will again re-form the correct base pairs, in a process is termed annealing or hybridization.

The rate of annealing is dependent upon the nucleotide sequence of the two strands of DNA. back to the top

Analysis of DNA Structure

Chromatography: Several of the chromatographic techniques available for the characterization of proteins can also be applied to the

characterization of DNA. The most commonly used technique is HPLC (high performance liquid chromatography). Affinity chromatographic techniques also

can be employed. One common affinity matrix is hydroxyapatite (a form of calcium phosphate), which binds double-stranded DNA with a higher affinity than

single-stranded DNA. back to the top

Electrophoresis: This procedure can serve the same function with regard to DNA molecules as it does for the analysis of proteins. However, since

DNA molecules have much higher molecular weights than proteins, the molecular sieve used in electrophoresis of DNA must be different as well. The

material of choice is agarose, a carbohydrate polymer purified from a salt water algae. It is a copolymer of mannose and galactose that when melted and re-

cooled forms a gel with pores sizes dependent upon the concentration of agarose. The phosphate backbone of DNA is highly negatively charged,

therefore DNA will migrate in an electric field. The size of DNA fragments can then be determined by comparing their migration in the gel to known size

standards. Extremely large molecules of DNA (in excess of 106 base pairs) are effectively separated in agarose gels using pulsed-field gel electrophoresis (PFGE). This technique employs two or more electrodes, placed orthogonally with respect to the gel, that receive short alternating pulses of current. PFGE

allows whole chromosomes and large portions of chromosomes to be analyzed. back to the top





• Primary Structure of Proteins • Secondary Structure of Proteins • Tertiary Structure of Proteins • Forces Controlling Structure • Quaternary Structure • Complex Protein Structures • Clinical Significances • Analysis of Protein Structure

o N-Terminal Analysis of Proteins o Protease Digestion for Peptide Generation o C-Terminal Analysis of Proteins o Chemical Digestion of Proteins o Size Exclusion Chromatography o Ion Exchange Chromatography o Affinity Chromatohgraphy

o High Performance/(Pressure) Liquid Chromatography o Electrophoresis of Proteins o Centrifugation of Proteins

• Myoglobin and Hemoglobin


Protein Primary Structure

The primary structure of peptides and proteins refers to the linear number and order of the amino acids present. The convention for the designation of the order of amino acids is that the N-terminal end (i.e. the end bearing the residue with the free α-amino group) is to the left (and the number 1 amino acid) and the C-terminal end (i.e. the end with the residue containing a free α-carboxyl group) is to the right. back to the top

Protein Secondary Structure

The ordered array of amino acids in a protein confer regular conformational forms upon that protein. These conformations constitute the secondary structures of a protein. In general proteins fold into two broad classes of structure termed, globular proteins or fibrous proteins. Globular proteins are compactly folded and coiled, whereas, fibrous proteins are more filamentous or elongated. It is the partial double-bond character of the peptide bond that defines the conformations a polypeptide chain may assume. Within a single protein different regions of the polypeptide chain may assume different conformations determined by the primary sequence of the amino acids.

The Alpha-Helix

The α-helix is a common secondary structure encountered in proteins of the globular class. The formation of the α-helix is spontaneous and is stabilized by H-bonding between amide nitrogens and carbonyl carbons of peptide bonds spaced four residues apart. This orientation of H-bonding produces a helical coiling of the peptide backbone such that the R-groups lie on the exterior of the helix and perpendicular to its axis. Not all amino acids favor the formation of the α-helix due to steric constraints of the R-groups. Amino acids such as A, D, E, I, L and M favor the formation of α-helices, whereas, G and P favor disruption of the helix. This is particularly true for P since it is a pyrrolidine based imino acid (HN=) whose structure significantly restricts movement about the peptide bond in which it is present, thereby, interfering with extension of the helix. The disruption of the helix is important as it

introduces additional folding of the polypeptide backbone to allow the formation of globular proteins.

ββββ -Sheets

Whereas an α-helix is composed of a single linear array of helically disposed amino acids, β-sheets are composed of 2 or more different regions of stretches of at least 5-10 amino acids. The folding and alignment of stretches of the polypeptide backbone aside one another to form β-sheets is stabilized by H-bonding between amide nitrogens and carbonyl carbons. However, the H-bonding residues are present in adjacently opposed stretches of the polypetide backbone as opposed to a linearly contiguous region of the backbone in the α-helix. β-Sheets are said to be pleated. This is due to positioning of the α-carbons of the peptide bond which alternates above and below the plane of the sheet. β-Sheets are either parallel or antiparallel. In parallel sheets adjacent peptide chains proceed in the same direction (i.e. the direction of N-terminal to C-terminal ends is the same), whereas, in antiparallel sheets adjacent chains are aligned in opposite directions.

Super-secondary Structure

Some proteins contain an ordered organization of secondary structures that form distinct functional domains or structural motifs. Examples include the helix-turn-helix domain of bacterial proteins that regulate transcription and the leucine zipper, helix-loop-helix and zinc finger domains of eukaryotic transcriptional regulators. These domains are termed super-secondary structures. back to the top

Tertiary Structure

Tertiary structure refers to the complete three-dimensional structure of the polypeptide units of a given protein. Included in this description is the spatial relationship of different secondary structures to one another within a polypeptide chain and how these secondary structures themselves fold into the three-dimensional form of the protein. Secondary structures of proteins often constitute distinct domains. Therefore, tertiary structure also describes the relationship of different domains to one another within a protein. The interactions of different domains is governed by several forces: These include hydrogen bonding, hydrophobic interactions, electrostatic interactions and van der Waals forces. back to the top

Forces Controlling Protein Structure

Hydrogen Bonding:

Polypeptides contain numerous proton donors and acceptors both in their backbone and in the R-groups of the amino acids. The environment in which proteins are found also contains the ample H-bond donors and acceptors of the water molecule. H-bonding, therefore, occurs not only within and between polypeptide chains but with the surrounding aqueous medium.

Hydrophobic Forces:

Proteins are composed of amino acids that contain either hydrophilic or hydrophobic R-groups. It is the nature of the interaction of the different R-groups with the aqueous environment that plays the major role in shaping protein structure. The spontaneous folded state of globular proteins is a reflection of a balance between the opposing energetics of H-bonding between hydrophilic R-groups and the aqueous environment and the repulsion from the aqueous environment by the hydrophobic R-groups. The hydrophobicity of certain amino acid R-groups tends to drive them away from the exterior of proteins and into the interior. This driving force restricts the available conformations into which a protein may fold.

Electrostatic Forces:

Electrostatic forces are mainly of three types; charge-charge, charge-dipole and dipole-dipole. Typical charge-charge interactions that favor protein folding are those between oppositely charged R-groups such as K or R and D or E. A substantial component of the energy involved in protein folding is charge-dipole interactions. This refers to the interaction of ionized R-groups of amino acids with the dipole of the water molecule. The slight dipole moment that exist in the polar R-groups of amino acid also influences their interaction with water. It is, therefore, understandable that the majority of the amino acids found on the exterior surfaces of globular proteins contain charged or polar R-groups.

van der Waals Forces:

There are both attractive and repulsive van der Waals forces that control protein folding. Attractive van der Waals forces involve the interactions among induced dipoles that arise from fluctuations in the charge densities that occur between adjacent uncharged non-bonded atoms. Repulsive van der Waals forces involve the interactions that occur when uncharged non-bonded atoms come very close together but do not induce dipoles. The repulsion is the result of the electron-electron repulsion that occurs as two clouds of electrons begin to overlap. Although van der Waals forces are extremely weak, relative to other forces governing conformation, it is the huge number of such interactions that occur in large protein molecules that make them significant to the folding of proteins. back to the top

Quaternary Structure

Many proteins contain 2 or more different polypeptide chains that are held in association by the same non-covalent forces that stabilize the tertiary structures of proteins. Proteins with multiple polypetide chains are termed oligomeric proteins. The structure formed by monomer-monomer interaction in an oligomeric protein is known as quaternary structure. Oligomeric proteins can be composed of multiple identical polypeptide chains or multiple distinct polypeptide chains. Proteins with identical subunits are termed homooligomers. Proteins containing several distinct polypeptide chains are termed heterooligomers. Hemoglobin, the oxygen carrying protein of the blood, contains two α and two β subunits arranged with a quaternary structure in the form, α2β2. Hemoglobin is, therefore, a hetero-oligomeric protein. back to the top

Complex Protein Structures

Proteins also are found to be covalently conjugated with carbohydrates. These modifications occur following the synthesis (translation) of proteins and are, therefore, termed post-translational modifications. These forms of modification impart specialized functions upon the resultant proteins. Proteins covalently associated with carbohydrates are termed glycoproteins. Glycoproteins are of two classes, N-linked and O-linked, referring to the site of covalent attachment of the sugar moieties. N-linked sugars are attached to the amide nitrogen of the R-group of asparagine; O-linked sugars are attached to the hydroxyl groups of either serine or threonine and occasionally to the hydroxyl group of the modified amino acid, hydroxylysine. There are extremely important glycoproteins found on the surface of erythrocytes. It is the variability in the composition of the carbohydrate portions of many glycoproteins and glycolipids of erythrocytes that determines blood group specificities. There are at least 100 blood group determinants, most of which are due to carbohydrate differences. The most common blood groups, A, B, and O, are specified by the activity of specific gene products whose activities are to incorporate distinct sugar groups onto RBC membrane glycoshpingolipids as well as secreted glycoproteins. Structural complexes involving protein associated with lipid via noncovalent interactions are termed lipoproteins. The distinct roles of lipoproteins are described on the linked page. Their major function in the body is to aid in the storage transport of lipid and cholesterol. back to the top

Clinical Significances

Visit the Inborn Errors page for a more complete listing of diseases related to abnormal proteins. Several brief examples are presented below. The substitution of a hydrophobic amino acid (V) for an acidic amino acid (E) in the β-chain of hemoglobin results in sickle cell anemia (HbS). This change of a single amino acid alters the structure of hemoglobin molecules in such a way that

the deoxygenated proteins polymerize and precipitate within the erythrocyte, leading to their characteristic sickle shape. Collagens are the most abundant proteins in the body. Alterations in collagen structure arising from abnormal genes or abnormal processing of collagen proteins results in numerous diseases, including Larsen syndrome, scurvy, osteogenesis imperfecta and Ehlers-Danlos syndrome. Ehlers-Danlos syndrome (see OMIM links) is actually the name associated with at least ten distinct disorders that are biochemically and clinically distinct yet all manifest structural weakness in connective tissue as a result of defective collagen structure. Osteogenesis imperfecta (see OMIM links) also encompasses more than one disorder. At least four biochemically and clinically distinguishable maladies have been identified as osteogenesis imperfecta, all of which are characterized by multiple fractures and resultant bone deformities. Marfan's syndrome manifests itself as a disorder of the connective tissue and was originally believed to be the result of abnormal collagens. However, recent evidence has shown that Marfan's syndrome results from mutations in the extracellular protein, fibrillin, which is an integral constituent of the non-collagenous microfibrils of the extracellular matrix. Several forms of familial hypercholesterolemia (see also OMIM links) are the result of genetic defects in the gene encoding the receptor for low-density lipoprotein (LDL). These defects result in the synthesis of abnormal LDL receptors that are incapable of binding to LDLs, or that bind LDLs but the receptor/LDL complexes are not properly internalized and degraded. The outcome is an elevation in serum cholesterol levels and increased propensity toward the development of atherosclerosis. A number of proteins can contribute to cellular transformation and carcinogenesis when their basic structure is disrupted by mutations in their genes. These genes are termed proto-oncogenes. For some of these proteins, all that is required to convert them to the oncogenic form is a single amino acid substitution. The cellular gene, c-Ras, is observed to sustain single amino acid substitutions at positions 12 or 61 with high frequency in colon carcinomas. Mutations in c-Ras are most frequently observed genetic alterations in colon cancer. back to the top

Amino-Terminal Sequence Determination

Prior to sequencing peptides it is necessary to eliminate disulfide bonds within peptides and between peptides. Several different chemical reactions can be used in order to permit separation of peptide strands and prevent protein conformations that are dependent upon disulfide bonds. The most common treatments are to use either 2-mercaptoethanol or dithiothreitol. Both of these chemicals reduce disulfide bonds. To prevent reformation of the disulfide bonds the peptides are treated with iodoacetic acid in order to alkylate the free sulfhydryls. There are three major chemical techniques for sequencing peptides and proteins from the N-terminus. These are the Sanger, Dansyl chloride and Edman techniques.

Sanger's Reagent: This sequencing technique utilizes the compound, 2,4-dinitrofluorobenzene (DNF) which reacts with the N-terminal residue under alkaline conditions. The derivatized amino acid can be hydrolyzed and will be labeled with a dinitrobenzene group that imparts a yellow color to the amino acid. Separation of the modified amino acids (DNP-derivative) by electrophoresis and comparison with the migration of DNP-derivative standards allows for the identification of the N-terminal amino acid. Dansyl chloride: Like DNF, dansyl chloride reacts with the N-terminal residue under alkaline conditions. Analysis of the modified amino acids is carried out similarly to the Sanger method except that the dansylated amino acids are detected by fluorescence. This imparts a higher sensitivity into this technique over that of the Sanger method. Edman degradation: The utility of the Edman degradation technique is that it allows for additional amino acid sequence to be obtained from the N-terminus inward. Using this method it is possible to obtain the entire sequence of peptides. This method utilizes phenylisothiocyanate to react with the N-terminal residue under alkaline conditions. The resultant phenylthiocarbamyl derivatized amino acid is hydrolyzed in anhydrous acid. The hydrolysis reaction results in a rearrangement of the released N-terminal residue to a phenylthiohydantoin derivative. As in the Sanger and Dansyl chloride methods, the N-terminal residue is tagged with an identifiable marker, however, the added advantage of the Edman process is that the remainder of the peptide is intact. The entire sequence of reactions can be repeated over and over to obtain the sequences of the peptide. This process has subsequently been automated to allow rapid and efficient sequencing of even extremely small quantities of peptide. back to the top

Protease Digestion

Due to the limitations of the Edman degradation technique, peptides longer than around 50 residues can not be sequenced completely. The ability to obtain peptides of this length, from proteins of greater length, is facilitated by the use of enzymes, endopeptidases, that cleave at specific sites within the primary sequence of proteins. The resultant smaller peptides can be chromatographically separated and subjected to Edman degradation sequencing reactions.

Specificities of Several Endoproteases

Enzyme Source Specificity Additional Points

Trypsin Bovine pancreas

peptide bond C-terminal to R, K, but not if next to P

highly specific for positively charged residues

Chymotrypsin Bovine pancreas

peptide bond C-terminal to F, Y, W but not if next to P

prefers bulky hydrophobic residues, cleaves slowly at N, H, M, L

Elastase Bovine pancreas

peptide bond C-terminal to A, G, S, V, but not if next to P

Thermolysin Bacillus thermoproteolyticus

peptide bond N-terminal to I, M, F, W, Y, V, but not if next to P

prefers small neutral residues, can cleave at A, D, H, T

Pepsin Bovine gastric mucosa

peptide bond N-terminal to L, F, W, Y, but when next to P

exhibits little specificity, requires low pH

Endopeptidase V8

Staphylococcus aureus

peptide bond C-terminal to E

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Carboxy-Terminal Sequence Determination

No reliable chemical techniques exist for sequencing the C-terminal amino acid of peptides. However, there are enzymes, exopeptidases, that have been identified that cleave peptides at the C-terminal residue which can then be analyzed chromatographically and compared to standard amino acids. This class of exopeptidases are called, carboxypeptidases.

Specificities of Several Exopeptidases

Enzyme Source Specificity

Carboxypeptidase A

Bovine pancreas

Will not cleave when C-terminal residue = R, K or P or if P resides next to terminal residue

Carboxypeptidase B

Bovine pancreas

Cleaves when C-terminal residue = R or K; not when P resides next to terminal reside

Carboxypeptidase C

Citrus leaves

All free C-terminal residues, pH optimum = 3.5

Carboxypeptidase Y Yeast All free C-terminal residues, slowly at G

residues

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Chemical Digestion of Proteins

The most commonly utilized chemical reagent that cleaves peptide bonds by recognition of specific amino acid residues is cyanogen bromide (CNBr). This reagent causes specific cleavage at the C-terminal side of M residues. The number of peptide fragments that result from CNBr cleavage is equivalent to one more than the number of M residues in a protein. The most reliable chemical technique for C-terminal residue identification is hydrazinolysis. A peptide is treated with hydrazine, NH2-NH2, at high temperature (90oC) for an extended length of time (20-100hr). This treatment cleaves all of the peptide bonds yielding amino-acyl hydrazides of all the amino acids excluding the C-terminal residue which can be identified chromatographically compared to amino acid standards. Due to the high percentage of hydrazine induced side reactions this technique is only used on carboxypeptidase resistant peptides. back to the top

Size Exclusion Chromatography

This chromatographic technique is based upon the use of a porous gel in the form of insoluble beads placed into a column. As a solution of proteins is passed through the column, small proteins can penetrate into the pores of the beads and, therefore, are retarded in their rate of travel through the column. The larger proteins a protein is the less likely it will enter the pores. Different beads with different pore sizes can be used depending upon the desired protein size separation profile. back to the top

Ion Exchange Chromatography Each individual protein exhibits a distinct overall net charge at a given pH. Some proteins will be negatively charged and some will be positively charged at the same pH. This property of proteins is the basis for ion exchange chromatography. Fine cellulose resins are used that are either negatively (cation exchanger) or positively (anion exchanger) charged. Proteins of opposite charge to the resin are retained as a solution of proteins is passed through the column. The bound proteins are then eluted by passing a solution of ions bearing a charge opposite to that of the column. By utilizing a gradient of increasing ionic strength, proteins with increasing affinity for the resin are progressively eluted. back to the top

Affinity Chromatography

Proteins have high affinities for their substrates or co-factors or prosthetic groups or receptors or antibodies raised against them. This affinity can be exploited in the purification of proteins. A column of beads bearing the high affinity compound can be prepared and a solution of protein passed through the column. The bound proteins are then eluted by passing a solution of unbound soluble high affinity compound through the column. back to the top

High Performance Liquid Chromatography (HPLC) In column chromatography the smaller and more tightly packed a resin is the greater the separation capability of the column. In gravity flow columns the limitation column packing is the time it takes to pass the solution of proteins through the column. HPLC utilizes tightly packed fine diameter resins to impart increased resolution and overcomes the flow limitations by pumping the solution of proteins through the column under high pressure. Like standard column chromatography, HPLC columns can be used for size exclusion or charge separation. An additional separation technique commonly used with HPLC is to utilize hydrophobic resins to retard the movement of nonpolar proteins. The proteins are then eluted from the column with a gradient of increasing concentration of an organic solvent. This latter form of HPLC is termed reversed-phase HPLC. back to the top

Electrophoresis of Proteins

Proteins also can be characterized according to size and charge by separation in an electric current (electrophoresis) within solid sieving gels made from polymerized and cross-linked acrylamide. The most commonly used technique is termed SDS polyacrylamide gel electrophoresis (SDS-PAGE). The gel is a thin slab of acrylamide polymerized between two glass plates. This technique utilizes a negatively charged detergent (sodium dodecyl sulfate) to denature and solubilize proteins. SDS denatured proteins have a uniform negative charge such

that all proteins will migrate through the gel in the electric field based solely upon size. The larger the protein the more slowly it will move through the matrix of the polyacrylamide. Following electrophoresis the migration distance of unknown proteins relative to known standard proteins is assessed by various staining or radiographic detection techniques. The use of polyacrylamide gel electrophoresis also can be used to determine the isoelectric charge of proteins (pI). This technique is termed isoelectric focusing. Isoelectric focusing utilizes a thin tube of polyacrylamide made in the presence of a mixture of small positively and negatively charged molecules termed ampholytes. The ampholytes have a range of pIs that establish a pH gradient along the gel when current is applied. Proteins will, therefore, cease migration in the gel when they reach the point where the ampholytes have established a pH equal to the proteins pI. back to the top

Centrifugation of Proteins

Proteins will sediment through a solution in a centrifugal field dependent upon their mass. Analytical centrifugation measure the rate that proteins sediment. The most common solution utilized is a linear gradient of sucrose (generally from 5-20%). Proteins are layered atop the gradient in an ultracentrifuge tube then subjected to centrifugal fields in excess of 100,000 x g. The sizes of unknown proteins can then be determined by comparing their migration distance in the gradient with those of known standard proteins. back to the top




Myoglobin Hemoglobin Role of 2,3-BPG


Myoglobin

Myoglobin and hemoglobin are hemeproteins whose physiological importance is principally related to their ability to bind molecular oxygen. Myoglobin is a monomeric heme protein found mainly in muscle tissue where it serves as an intracellular storage site for oxygen. During periods of oxygen deprivation oxymyoglobin releases its bound oxygen which is then used for metabolic purposes. The tertiary structure of myoglobin is that of a typical water soluble globular protein. Its secondary structure is unusual in that it contains a very high proportion (75%) of α-helical secondary structure. A myoglobin polypeptide is comprised of 8 separate right handed α-helices, designated A through H, that are connected by short non helical regions. Amino acid R-groups packed into the interior of the molecule are predominantly hydrophobic in character while those exposed on the surface of the molecule are generally hydrophilic, thus making the molecule relatively water soluble. Each myoglobin molecule contains one heme prosthetic group inserted into a hydrophobic cleft in the protein. Each heme residue contains one central coordinately bound iron atom that is normally in the Fe2+, or ferrous, oxidation state. The oxygen carried by hemeproteins is bound directly to the ferrous iron atom of the heme prosthetic group. Oxidation of the iron to the Fe3+, ferric, oxidation state renders the molecule incapable of normal oxygen binding. Hydrophobic interactions between the tetrapyrrole ring and hydrophobic amino acid R groups on the interior of the cleft in the protein strongly stabilize the heme protein conjugate. In addition a nitrogen atom from a histidine R group located above the plane of the heme ring is coordinated with the iron atom further stabilizing the interaction between the heme and the protein. In oxymyoglobin the remaining bonding site on the iron atom (the 6th coordinate position) is occupied by the oxygen, whose binding is stabilized by a second histidine residue. Carbon monoxide also binds coordinately to heme iron atoms in a manner similar to that of oxygen, but the binding of carbon monoxide to heme is much stronger than that of oxygen. The preferential binding of carbon monoxide to heme iron is largely responsible for the asphyxiation that results from carbon monoxide poisoning. back to the top

Hemoglobin Hemoglobin is an [α(2):β(2)] tetrameric hemeprotein found in erythrocytes where it is responsible for binding oxygen in the lung and transporting the bound oxygen throughout the body where it is used in aerobic metabolic pathways. Each subunit of a hemoglobin tetramer has a heme prosthetic group identical to that described for myoglobin. The common peptide subunits are designated α, β, γ and δ which are arranged into the most commonly occurring functional hemoglobins. Although the secondary and tertiary structure of various hemoglobin subunits are similar, reflecting extensive homology in amino acid composition, the variations in amino acid composition that do exist impart marked differences in hemoglobin's oxygen carrying properties. In addition, the quaternary structure of hemoglobin leads to physiologically important allosteric interactions between the subunits, a property lacking in monomeric myoglobin which is otherwise very similar to the α-subunit of hemoglobin. Comparison of the oxygen binding properties of myoglobin and hemoglobin illustrate the allosteric properties of hemoglobin that results from its quaternary structure and differentiate hemoglobin's oxygen binding properties from that of myoglobin. The curve of oxygen binding to hemoglobin is sigmoidal typical of allosteric proteins in which the substrate, in this case oxygen, is a positive homotropic effector. When oxygen binds to the first subunit of deoxyhemoglobin it increases the affinity of the remaining subunits for oxygen. As additional oxygen is bound to the second and third subunits oxygen binding is further, incrementally, strengthened, so that at the oxygen tension in lung alveoli, hemoglobin is fully saturated with oxygen. As oxyhemoglobin circulates to deoxygenated tissue, oxygen is incrementally unloaded and the affinity of hemoglobin for oxygen is reduced. Thus at the lowest oxygen tensions found in very active tissues the binding affinity of hemoglobin for oxygen is very low allowing maximal delivery of oxygen to the tissue. In contrast the oxygen binding curve for myoglobin is hyperbolic in character indicating the absence of allosteric interactions in this process. The allosteric oxygen binding properties of hemoglobin arise directly from the interaction of oxygen with the iron atom of the heme prosthetic groups and the resultant effects of these interactions on the quaternary structure of the protein. When oxygen binds to an iron atom of deoxyhemoglobin it pulls the iron atom into the plane of the heme. Since the iron is also bound to histidine F8, this residue is also pulled toward the plane of the heme ring. The conformational change at histidine F8 is transmitted throughout the peptide backbone resulting in a significant change in tertiary structure of the entire subunit. Conformational changes at the subunit surface lead to a new set of binding interactions between adjacent subunits. The latter changes include disruption of salt bridges and formation of new hydrogen bonds and new hydrophobic interactions, all of which contribute to the new quaternary structure. The latter changes in subunit interaction are transmitted, from the surface, to the heme binding pocket of a second deoxy subunit and result in easier access of oxygen to the iron atom of the second heme and thus a greater affinity of the

hemoglobin molecule for a second oxygen molecule. The tertiary configuration of low affinity, deoxygenated hemoglobin (Hb) is known as the taut (T) state. Conversely, the quaternary structure of the fully oxygenated high affinity form of hemoglobin (HbO2) is known as the relaxed (R) state. In the context of the affinity of hemoglobin for oxygen there are four primary regulators, each of which has a negative impact. These are CO2, hydrogen ion (H+), chloride ion (Cl-), and 2,3-bisphosphoglycerate (2,3BPG, or also just BPG). Some older texts abbreviate 2,3BPG as DPB. Although they can influence O2 binding independent of each other, CO2, H+ and Cl- primarily function as a consequence of each other on the affinity of hemoglobin for O2. We shall consider the transport of O2 from the lungs to the tissues first. In the high O2 environment (high pO2) of the lungs there is sufficient O2 to overcome the inhibitory nature of the T state. During the O2 binding-induced alteration from the T form to the R form several amino acid side groups on the surface of hemoglobin subunits will dissociate protons as depicted in the equation below. This proton dissociation plays an important role in the expiration of the CO2 that arrives from the tissues (see below). However, because of the high pO2, the pH of the blood in the lungs (~7.4 - 7.5) is not sufficiently low enough to exert a negative influence on hemoglobin binding O2. When the oxyhemoglobin reaches the tissues the pO2 is sufficiently low, as well as the pH (~7.2), that the T state is favored and the O2 released.

4O2 + Hb <--------> nH+ + Hb(O2)4 If we now consider what happens in the tissues, it is possible to see how CO2, H+ and Cl- exert their negative effects on hemoglobin binding O2. Metabolizing cells produce CO2 which diffuses into the blood and enters the circulating red blood cells (RBCs). Within RBCs the CO2 is rapidly converted to carbonic acid through the action of carbonic anhydrase as shown in the equation below:

CO2 + H2O --------> H2CO3 ------> H+ + HCO3-

The bicarbonate ion produced in this dissociation reaction diffuses out of the RBC and is carried in the blood to the lungs. This effective CO2 transport process is referred to as isohydric transport. Approximately 80% of the CO2 produced in metabolizing cells is transported to the lungs in this way. A small percentage of CO2 is transported in the blood as a dissolved gas. In the tissues, the H+ dissociated from carbonic acid is buffered by hemoglobin which exerts a negative influence on O2 binding forcing release to the tissues. As indicated above, within the lungs the high pO2 allows for effective O2 binding by hemoglobin leading to the T to R state transition and the release of protons. The protons combine with the bicarbonate that arrived from the tissues forming carbonic acid which then enters the RBCs. Through a reversal of the carbonic anhydrase reaction, CO2 and H2O are produced. The CO2 diffuses out of the blood, into the lung alveoli and is released on expiration. In addition to isohydric transport, as much as 15% of CO2 is transported to the lungs bound to N-terminal amino groups of the T form of hemoglobin. This reaction, depicted below, forms what is called carbamino-hemoglobin. As indicated this reaction also produces H+, thereby lowering the pH in tissues where the CO2 concentration is high. The formation of H+ leads to release of the

bound O2 to the surrounding tissues. Within the lungs, the high O2 content results in O2 binding to hemoglobin with the concomitant release of H+. The released protons then promote the dissociation of the carbamino to form CO2 which is then released with expiration.

CO2 + Hb-NH2 <-----> H+ + Hb-NH-COO- As the above discussion demonstrates, the conformation of hemoglobin and its oxygen binding are sensitive to hydrogen ion concentration. These effects of hydrogen ion concentration are responsible for the well known Bohr effect in which increases in hydrogen ion concentration decrease the amount of oxygen bound by hemoglobin at any oxygen concentration (partial pressure). Coupled to the diffusion of bicarbonate out of RBCs in the tissues there must be ion movement into the RBCs to maintain electrical neutrality. This is the role of Cl- and is referred to as the chloride shift. In this way, Cl- plays an important role in bicarbonate production and diffusion and thus also negatively influences O2 binding to hemoglobin.

Representation of the transport of CO2 from the tissues to the blood with delivery of O2 to the tissues. The opposite process occurs when O2 is taken up from the alveoli of the lungs and the CO2 is expelled. All of the processes of the transport of CO2 and O2 are not shown such as the formation and ionization of carbonic acid in the plasma. The latter is a major mechanism for the transport of CO2 to the lungs, i.e. in the plasma as HCO3

-. The H+ produced in the plasma by the

ionization of carbonic acid is buffered by phosphate (HPO42-) and by

proteins. Additionally, some 15% of the CO2 is transported from the tissues to the lungs as hemoglobin carbamate.

back to the top

Role of 2,3-bisphosphoglycerate (2,3-BPG)

The compound 2,3-bisphosphoglycerate (2,3-BPG), derived from the glycolytic intermediate 1,3-bisphosphoglycerate, is a potent allosteric effector on the oxygen binding properties of hemoglobin. The formation of 2,3-BPG is diagrammed. In the deoxygenated T conformer, a cavity capable of binding 2,3-BPG forms in the center of the molecule. 2,3-BPG can occupy this cavity stabilizing the T state. Conversely, when 2,3-BPG is not available, or not bound in the central cavity, Hb can be converted to HbO2 more readily. Thus, like increased hydrogen ion concentration, increased 2,3-BPG concentration favors conversion of R form Hb to T form Hb and decreases the amount of oxygen bound by Hb at any oxygen concentration. Hemoglobin molecules differing in subunit composition are known to have different 2,3-BPG binding properties with correspondingly different allosteric responses to 2,3-BPG. For example, HbF (the fetal form of hemoglobin) binds 2,3-BPG much less avidly than HbA (the adult form of hemoglobin) with the result that HbF in fetuses of pregnant women binds oxygen with greater affinity than the mothers HbA, thus giving the fetus preferential access to oxygen carried by the mothers circulatory system. back to the top

return to Protein Structure Page



Last modified: Thursday, 22-Jan-2004 10:02:47 EST

• Introduction to Enzymes • Enzyme Classifications • Role of Coenzymes

• Enzyme Activity Relative to Substrate Type • Enzyme-Substrate Interactions • Chemical Reactions and Rates • Chemical Reaction Order • Enzymes as Biological Catalysts • Michaelis-Menton Kinetics • Inhibition of Enzyme Catalyzed Reactions • Regulation of Enzyme Activity • Allosteric Enzymes • Enzymes in the Diagnosis of Pathology

Enzyme Kinetics by Dr. Peter Birch, University of Paisley


Introduction to Enzymes Enzymes are biological catalysts responsible for supporting almost all of the chemical reactions that maintain animal homeostasis. Because of their role in maintaining life processes, the assay and pharmacological regulation of enzymes have become key elements in clinical diagnosis and therapeutics. The macromolecular components of almost all enzymes are composed of protein, except for a class of RNA modifying catalysts known as ribozymes. Ribozymes are molecules of ribonucleic acid that catalyze reactions on the phosphodiester bond of other RNAs. Enzymes are found in all tissues and fluids of the body. Intracellular enzymes catalyze the reactions of metabolic pathways. Plasma membrane enzymes regulate catalysis within cells in response to extracellular signals, and enzymes of the circulatory system are responsible for regulating the clotting of blood. Almost every significant life process is dependent on enzyme activity. back to the top

Enzyme Classifications

Traditionally, enzymes were simply assigned names by the investigator who discovered the enzyme. As knowledge expanded, systems of enzyme classification became more comprehensive and complex. Currently enzymes are grouped into six functional classes by the International Union of Biochemists (I.U.B.).

Number Classification Biochemical Properties

1. Oxidoreductases Act on many chemical groupings to add or remove

hydrogen atoms.

2. Transferases

Transfer functional groups between donor and acceptor molecules. Kinases are specialized transferases that regulate metabolism by transferring phosphate from ATP to other molecules.

3. Hydrolases Add water across a bond, hydrolyzing it.

4. Lyases

Add water, ammonia or carbon dioxide across double bonds, or remove these elements to produce double bonds.

5. Isomerases

Carry out many kinds of isomerization: L to D isomerizations, mutase reactions (shifts of chemical groups) and others.

6. Ligases

Catalyze reactions in which two chemical groups are joined (or ligated) with the use of energy from ATP.

These rules give each enzyme a unique number. The I.U.B. system also

specifies a textual name for each enzyme. The enzyme's name is comprised of the names of the substrate(s), the product(s) and the enzyme's functional class. Because many enzymes, such as alcohol dehydrogenase, are widely known in

the scientific community by their common names, the change to I.U.B.-approved nomenclature has been slow. In everyday usage, most enzymes are still called

by their common name. Enzymes are also classified on the basis of their composition. Enzymes

composed wholly of protein are known as simple enzymes in contrast to complex enzymes, which are composed of protein plus a relatively small

organic molecule. Complex enzymes are also known as holoenzymes. In this terminology the protein component is known as the apoenzyme, while the non-

protein component is known as the coenzyme or prosthetic group where prosthetic group describes a complex in which the small organic molecule is

bound to the apoenzyme by covalent bonds; when the binding between the apoenzyme and non-protein components is non-covalent, the small organic molecule is called a coenzyme. Many prosthetic groups and coenzymes are water-soluble derivatives of vitamins. It should be noted that the main clinical

symptoms of dietary vitamin insufficiency generally arise from the malfunction of enzymes, which lack sufficient cofactors derived from vitamins to maintain

homeostasis. The non-protein component of an enzyme may be as simple as a metal ion or as complex as a small non-protein organic molecule. Enzymes that require a metal in their composition are known as metalloenzymes if they bind and retain their metal atom(s) under all conditions, that is with very high affinity. Those which have a lower affinity for metal ion, but still require the metal ion for activity, are

known as metal-activated enzymes. back to the top

Role of Coenzymes

The functional role of coenzymes is to act as transporters of chemical groups from one reactant to another. The chemical groups carried can be as simple as

the hydride ion (H+ + 2e-) carried by NAD or the mole of hydrogen carried by FAD; or they can be even more complex than the amine (-NH2) carried by

pyridoxal phosphate. Since coenzymes are chemically changed as a consequence of enzyme action, it

is often useful to consider coenzymes to be a special class of substrates, or second substrates, which are common to many different holoenzymes. In all cases, the coenzymes donate the carried chemical grouping to an acceptor

molecule and are thus regenerated to their original form. This regeneration of coenzyme and holoenzyme fulfills the definition of an enzyme as a chemical

catalyst, since (unlike the usual substrates, which are used up during the course of a reaction) coenzymes are generally regenerated.

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Enzyme Relative to Substrate Type Although enzymes are highly specific for the kind of reaction they catalyze, the same is not always true of substrates they attack. For example, while succinic

dehydrogenase (SDH) always catalyzes an oxidation-reduction reaction and its substrate is invariably succinic acid, alcohol dehydrogenase (ADH) always

catalyzes oxidation-reduction reactions but attacks a number of different alcohols, ranging from methanol to butanol. Generally, enzymes having broad

substrate specificity are most active against one particular substrate. In the case of ADH, ethanol is the preferred substrate.

Enzymes also are generally specific for a particular steric configuration (optical isomer) of a substrate. Enzymes that attack D sugars will not attack the

corresponding L isomer. Enzymes that act on L amino acids will not employ the corresponding D optical isomer as a substrate. The enzymes known as

racemases provide a striking exception to these generalities; in fact, the role of racemases is to convert D isomers to L isomers and vice versa. Thus racemases

attack both D and L forms of their substrate. As enzymes have a more or less broad range of substrate specificity, it follows that a given substrate may be acted on by a number of different enzymes, each

of which uses the same substrate(s) and produces the same product(s). The individual members of a set of enzymes sharing such characteristics are known

as isozymes. These are the products of genes that vary only slightly; often, various isozymes of a group are expressed in different tissues of the body. The best studied set of isozymes is the lactate dehydrogenase (LDH) system. LDH is a tetrameric enzyme composed of all possible arrangements of two different

protein subunits; the subunits are known as H (for heart) and M (for skeletal muscle). These subunits combine in various combinations leading to 5 distinct

isozymes. The all H isozyme is characteristic of that from heart tissue, and the all M isozyme is typically found in skeletal muscle and liver. These isozymes all

catalyze the same chemical reaction, but they exhibit differing degrees of efficiency. The detection of specific LDH isozymes in the blood is highly

diagnostic of tissue damage such as occurs during cardiac infarct. back to the top

Enzyme-Substrate Interactions

The favored model of enzyme substrate interaction is known as the induced fit model. This model proposes that the initial interaction between enzyme and substrate is relatively weak, but that these weak interactions rapidly induce

conformational changes in the enzyme that strengthen binding and bring catalytic sites close to substrate bonds to be altered. After binding takes place, one or

more mechanisms of catalysis generates transition- state complexes and reaction products. The possible mechanisms of catalysis are four in number: 1. Catalysis by Bond Strain: In this form of catalysis, the induced structural

rearrangements that take place with the binding of substrate and enzyme ultimately produce strained substrate bonds, which more easily attain the

transition state. The new conformation often forces substrate atoms and bulky catalytic groups, such as aspartate and glutamate, into conformations that strain

existing substrate bonds. 2. Catalysis by Proximity and Orientation: Enzyme-substrate interactions

orient reactive groups and bring them into proximity with one another. In addition to inducing strain, groups such as aspartate are frequently chemically reactive as

well, and their proximity and orientation toward the substrate thus favors their participation in catalysis.

3. Catalysis Involving Proton Donors (Acids) and Acceptors (Bases): Other mechanisms also contribute significantly to the completion of catalytic events

initiated by a strain mechanism, for example, the use of glutamate as a general acid catalyst (proton donor).

4. Covalent Catalysis: In catalysis that takes place by covalent mechanisms, the substrate is oriented to active sites on the enzymes in such a way that a

covalent intermediate forms between the enzyme or coenzyme and the substrate. One of the best-known examples of this mechanism is that involving

proteolysis by serine proteases, which include both digestive enzymes (trypsin, chymotrypsin, and elastase) and several enzymes of the blood clotting

cascade. These proteases contain an active site serine whose R group hydroxyl forms a covalent bond with a carbonyl carbon of a peptide bond, thereby causing

hydrolysis of the peptide bond. back to the top

Chemical Reactions and Rates

According to the conventions of biochemistry, the rate of a chemical reaction is described by the number of molecules of reactant(s) that are converted into

product(s) in a specified time period. Reaction rate is always dependent on the concentration of the chemicals involved in the process and on rate constants that

are characteristic of the reaction. For example, the reaction in which A is converted to B is written as follows:

A ------> B The rate of this reaction is expressed algebraically as either a decrease in the

concentration of reactant A: -[A] = k[B]

or an increase in the concentration of product B:

[B] = k[A] In the second equation (of the 3 above) the negative sign signifies a decrease in concentration of A as the reaction progresses, brackets define concentration in

molarity and the k is known as a rate constant. Rate constants are simply proportionality constants that provide a quantitative connection between chemical

concentrations and reaction rates. Each chemical reaction has characteristic values for its rate constants; these in turn directly relate to the equilibrium

constant for that reaction. Thus, reaction can be rewritten as an equilibrium expression in order to show the relationship between reaction rates, rate

constants and the equilibrium constant for this simple case. The rate constant for the forward reaction is defined as k+1 and the reverse as k-1.

At equilibrium the rate (v) of the forward reaction (A -----> B) is--- by definition--- equal to that of the reverse or back reaction (B -----> A), a relationship which is

algebraically symbolized as: vforward = vreverse

where, for the forward reaction:

vforward = k+1[A]

and for the reverse reaction:

vreverse = k-1[B] In the above equations, k+1 and k-1 represent rate constants for the forward and reverse reactions, respectively. The negative subscript refers only to a reverse

reaction, not to an actual negative value for the constant. To put the relationships of the two equations into words, we state that the rate of the forward reaction [vforward] is equal to the product of the forward rate constant k+1 and the molar

concentration of A. The rate of the reverse reaction is equal to the product of the reverse rate constant k-1 and the molar concentration of B.

At equilibrium, the rate of the forward reaction is equal to the rate of the reverse reaction leading to the equilibrium constant of the reaction and is expressed

by: [B]/[A] = k+1/k-1 = Keq

This equation demonstrates that the equilibrium constant for a chemical reaction is not only equal to the equilibrium ratio of product and reactant concentrations, but is also equal to the ratio of the characteristic rate constants of the reaction.

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Chemical Reaction Order

Reaction order refers to the number of molecules involved in forming a reaction complex that is competent to proceed to product(s). Empirically, order is easily determined by summing the exponents of each concentration term in the rate

equation for a reaction. A reaction characterized by the conversion of one molecule of A to one molecule of B with no influence from any other reactant or solvent is a first-order reaction. The exponent on the substrate concentration in

the rate equation for this type of reaction is 1. A reaction with two substrates forming two products would a second-order reaction. However, the reactants in second- and higher- order reactions need not be different chemical species. An

example of a second order reaction is the formation of ATP through the condensation of ADP with orthophosphate:

ADP + H2PO4 <----> ATP + H2O

For this reaction the forward reaction rate would be written as:

vforward = k1[ADP][H2PO4]

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Enzymes as Biological Catalysts

In cells and organisms most reactions are catalyzed by enzymes, which are regenerated during the course of a reaction. These biological catalysts are

physiologically important because they speed up the rates of reactions that would otherwise be too slow to support life. Enzymes increase reaction rates---

sometimes by as much as one millionfold, but more typically by about one thousand fold. Catalysts speed up the forward and reverse reactions

proportionately so that, although the magnitude of the rate constants of the forward and reverse reactions is are increased, the ratio of the rate constants

remains the same in the presence or absence of enzyme. Since the equilibrium constant is equal to a ratio of rate constants, it is apparent that enzymes and

other catalysts have no effect on the equilibrium constant of the reactions they catalyze.

Enzymes increase reaction rates by decreasing the amount of energy required to form a complex of reactants that is competent to produce reaction products. This

complex is known as the activated state or transition state complex for the reaction. Enzymes and other catalysts accelerate reactions by lowering the energy of the transition state. The free energy required to form an activated

complex is much lower in the catalyzed reaction. The amount of energy required to achieve the transition state is lowered; consequently, at any instant a greater proportion of the molecules in the population can achieve the transition state.

The result is that the reaction rate is increased. back to the top

Michaelis-Menton Kinetics

In typical enzyme-catalyzed reactions, reactant and product concentrations are usually hundreds or thousands of times greater than the enzyme concentration.

Consequently, each enzyme molecule catalyzes the conversion to product of many reactant molecules. In biochemical reactions, reactants are commonly known as substrates. The catalytic event that converts substrate to product

involves the formation of a transition state, and it occurs most easily at a specific binding site on the enzyme. This site, called the catalytic site of the enzyme, has been evolutionarily structured to provide specific, high-affinity binding of

substrate(s) and to provide an environment that favors the catalytic events. The complex that forms when substrate(s) and enzyme combine is called the enzyme substrate (ES) complex. Reaction products arise when the ES complex breaks

down releasing free enzyme. Between the binding of substrate to enzyme, and the reappearance of free

enzyme and product, a series of complex events must take place. At a minimum an ES complex must be formed; this complex must pass to the transition state (ES*); and the transition state complex must advance to an enzyme product

complex (EP). The latter is finally competent to dissociate to product and free enzyme. The series of events can be shown thus:

E + S <---> ES <---> ES* <---> EP <---> E + P The kinetics of simple reactions like that above were first characterized by

biochemists Michaelis and Menten. The concepts underlying their analysis of enzyme kinetics continue to provide the cornerstone for understanding

metabolism today, and for the development and clinical use of drugs aimed at selectively altering rate constants and interfering with the progress of disease

states. The Michaelis-Menten equation:

is a quantitative description of the relationship among the rate of an enzyme- catalyzed reaction [v1], the concentration of substrate [S] and two constants, Vmax

and Km (which are set by the particular equation). The symbols used in the Michaelis-Menton equation refer to the reaction rate [v1], maximum reaction rate

(Vmax), substrate concentration [S] and the Michaelis-Menton constant (Km).

The Michaelis-Menten equation can be used to demonstrate that at the substrate concentration that produces exactly half of the maximum reaction rate, i.e.,1/2

Vmax, the substrate concentration is numerically equal to Km. This fact provides a simple yet powerful bioanalytical tool that has been used to characterize both

normal and altered enzymes, such as those that produce the symptoms of genetic diseases. Rearranging the Michaelis-Menton equation leads to:

From this equation it should be apparent that when the substrate concentration is half that required to support the maximum rate of reaction, the observed rate, v1, will, be equal to Vmax divided by 2; in other words, v1 = [Vmax/2]. At this substrate

concentration Vmax/v1 will be exactly equal to 2, with the result that [S](1) = Km

The latter is an algebraic statement of the fact that, for enzymes of the Michaelis-Menten type, when the observed reaction rate is half of the maximum possible reaction rate, the substrate concentration is numerically equal to the Michaelis-Menten constant. In this derivation, the units of Km are those used to specify the

concentration of S, usually Molarity. The Michaelis-Menten equation has the same form as the equation for a

rectangular hyperbola; graphical analysis of reaction rate (v) versus substrate concentration [S] produces a hyperbolic rate plot.

Plot of substrate concentration versus reaction velocity

The key features of the plot are marked by points A, B and C. At high substrate concentrations the rate represented by point C the rate of the reaction is almost equal to Vmax, and the difference in rate at nearby concentrations of substrate is almost negligible. If the Michaelis-Menten plot is extrapolated to infinitely high

substrate concentrations, the extrapolated rate is equal to Vmax. When the reaction rate becomes independent of substrate concentration, or nearly so, the

rate is said to be zero order. (Note that the reaction is zero order only with respect to this substrate. If the reaction has two substrates, it may or may not be zero order with respect to the second substrate). The very small differences in reaction velocity at substrate concentrations around point C (near Vmax) reflect the fact that at these concentrations almost all of the enzyme molecules are

bound to substrate and the rate is virtually independent of substrate, hence zero order. At lower substrate concentrations, such as at points A and B, the lower reaction velocities indicate that at any moment only a portion of the enzyme molecules are bound to the substrate. In fact, at the substrate concentration

denoted by point B, exactly half the enzyme molecules are in an ES complex at any instant and the rate is exactly one half of Vmax. At substrate concentrations

near point A the rate appears to be directly proportional to substrate concentration, and the reaction rate is said to be first order.

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Inhibition of Enzyme Catalyzed Reactions

To avoid dealing with curvilinear plots of enzyme catalyzed reactions, biochemists Lineweaver and Burk introduced an analysis of enzyme kinetics

based on the following rearrangement of the Michaelis-Menten equation: [1/v] = [Km (1)/ Vmax[S] + (1)/Vmax]

Plots of 1/v versus 1/[S] yield straight lines having a slope of Km/Vmax and an intercept on the ordinate at 1/Vmax.

A Lineweaver-Burk Plot

An alternative linear transformation of the Michaelis-Menten equation is the Eadie-Hofstee transformation:

v/[S] = -v [1/Km] + [Vmax/Km]

and when v/[S] is plotted on the y-axis versus v on the x-axis, the result is a linear plot with a slope of -1/Km and the value Vmax/Km as the intercept on the y-

axis and Vmax as the intercept on the x-axis.

Both the Lineweaver-Burk and Eadie-Hofstee transformation of the Michaelis-Menton equation are useful in the analysis of enzyme inhibition. Since most

clinical drug therapy is based on inhibiting the activity of enzymes, analysis of enzyme reactions using the tools described above has been fundamental to the

modern design of pharmaceuticals . Well- known examples of such therapy include the use of methotrexate in cancer chemotherapy to semi-selectively

inhibit DNA synthesis of malignant cells, the use of aspirin to inhibit the synthesis of prostaglandins which are at least partly responsible for the aches and pains of

arthritis, and the use of sulfa drugs to inhibit the folic acid synthesis that is essential for the metabolism and growth of disease-causing bacteria. In addition,

many poisons--- such as cyanide, carbon monoxide and polychlorinated

biphenols (PCBs)--- produce their life- threatening effects by means of enzyme inhibition.

Enzyme inhibitors fall into two broad classes: those causing irreversible inactivation of enzymes and those whose inhibitory effects can be reversed.

Inhibitors of the first class usually cause an inactivating, covalent modification of enzyme structure. Cyanide is a classic example of an irreversible enzyme

inhibitor: by covalently binding mitochondrial cytochrome oxidase, it inhibits all the reactions associated with electron transport. The kinetic effect of irreversible inhibitors is to decrease the concentration of active enzyme, thus decreasing the

maximum possible concentration of ES complex. Since the limiting enzyme reaction rate is often k2[ES], it is clear that under these circumstances the

reduction of enzyme concentration will lead to decreased reaction rates. Note that when enzymes in cells are only partially inhibited by irreversible inhibitors, the remaining unmodified enzyme molecules are not distinguishable from those

in untreated cells; in particular, they have the same turnover number and the same Km. Turnover number, related to Vmax, is defined as the maximum number of moles of substrate that can be converted to product per mole of catalytic site per second. Irreversible inhibitors are usually considered to be poisons and are

generally unsuitable for therapeutic purposes. Reversible inhibitors can be divided into two main categories--- competitive

inhibitors and noncompetitive inhibitors---with a third category, uncompetitive inhibitors, rarely encountered.

Inhibitor Type

Binding Site on Enzyme

Kinetic effect

Competitive Inhibitor

Specifically at the catalytic site, where it competes with substrate for binding in a dynamic equilibrium- like process. Inhibition is reversible by substrate.

Vmax is unchanged; Km, as defined by [S] required for 1/2 maximal activity, is increased.

Noncompetitive Inhibitor

Binds E or ES complex other than at the catalytic site. Substrate binding unaltered, but ESI complex cannot form products. Inhibition cannot be reversed by substrate.

Km appears unaltered; Vmax is decreased proportionately to inhibitor concentration.

Uncompetitive Inhibitor

Binds only to ES complexes at locations other than the catalytic site. Substrate binding modifies enzyme structure, making inhibitor- binding site available. Inhibition cannot be reversed by substrate.

Apparent Vmax decreased; Km, as defined by [S] required for 1/2 maximal activity, is decreased.

The hallmark of all the reversible inhibitors is that when the inhibitor

concentration drops, enzyme activity is regenerated. Usually these inhibitors bind to enzymes by non-covalent forces and the inhibitor maintains a reversible

equilibrium with the enzyme. The equilibrium constant for the dissociation of enzyme inhibitor complexes is known as KI:

KI = [E][I]/[E--I--complex] The importance of KI is that in all enzyme reactions where substrate, inhibitor and

enzyme interact, the normal Km and or Vmax for substrate enzyme interaction appear to be altered. These changes are a consequence of the influence of KI on the overall rate equation for the reaction. The effects of KI are best observed in

Lineweaver-Burk plots. Probably the best known reversible inhibitors are competitive inhibitors, which always bind at the catalytic or active site of the enzyme. Most drugs that alter

enzyme activity are of this type. Competitive inhibitors are especially attractive as clinical modulators of enzyme activity because they offer two routes for the

reversal of enzyme inhibition, while other reversible inhibitors offer only one. First, as with all kinds of reversible inhibitors, a decreasing concentration of the

inhibitor reverses the equilibrium, regenerating active free enzyme. Second, since substrate and competitive inhibitors both bind at the same site, they

compete with one another for binding Raising the concentration of substrate (S), while holding the concentration of

inhibitor constant, provides the second route for reversal of competitive inhibition. The greater the proportion of substrate, the greater the proportion of enzyme

present in competent ES complexes. As noted earlier, high concentrations of substrate can displace virtually all

competitive inhibitor bound to active sites. Thus, it is apparent that Vmax should be unchanged by competitive inhibitors. This characteristic of competitive

inhibitors is reflected in the identical vertical-axis intercepts of Lineweaver-Burk plots, with and without inhibitor.

Lineweaver-Burk Plots of Inhibited Enzymes

Since attaining Vmax requires appreciably higher substrate concentrations in the presence of competitive inhibitor, Km (the substrate concentration at half maximal velocity) is also higher, as demonstrated by the differing negative intercepts on

the horizontal axis in panel B. Analogously, panel C illustrates that noncompetitive inhibitors appear to have no effect on the intercept at the x-axis implying that noncompetitive inhibitors have no effect on the Km of the enzymes they inhibit. Since noncompetitive inhibitors do not interfere in the equilibration of enzyme, substrate and ES complexes, the

Km's of Michaelis-Menten type enzymes are not expected to be affected by noncompetitive inhibitors, as demonstrated by x-axis intercepts in panel C. However, because complexes that contain inhibitor (ESI) are incapable of

progressing to reaction products, the effect of a noncompetitive inhibitor is to reduce the concentration of ES complexes that can advance to product. Since Vmax = k2[Etotal], and the concentration of competent Etotal is diminished by the

amount of ESI formed, noncompetitive inhibitors are expected to decrease Vmax, as illustrated by the y-axis intercepts in panel C.

A corresponding analysis of uncompetitive inhibition leads to the expectation that these inhibitors should change the apparent values of Km as well as Vmax.

Changing both constants leads to double reciprocal plots, in which intercepts on the x and y axes are proportionately changed; this leads to the production of

parallel lines in inhibited and uninhibited reactions. back to the top

Regulation of Enzyme Activity

While it is clear that enzymes are responsible for the catalysis of almost all biochemical reactions, it is important to also recognize that rarely, if ever, do enzymatic reactions proceed in isolation. The most common scenario is that

enzymes catalyze individual steps of multi-step metabolic pathways, as is the case with glycolysis, gluconeogenesis or the synthesis of fatty acids. As a

consequence of these lock- step sequences of reactions, any given enzyme is dependent on the activity of preceding reaction steps for its substrate.

In humans, substrate concentration is dependent on food supply and is not usually a physiologically important mechanism for the routine regulation of

enzyme activity. Enzyme concentration, by contrast, is continually modulated in response to physiological needs. Three principal mechanisms are known to

regulate the concentration of active enzyme in tissues:

• 1. Regulation of gene expression controls the quantity and rate of enzyme synthesis.

• 2. Proteolytic enzyme activity determines the rate of enzyme degradation. • 3. Covalent modification of preexisting pools of inactive proenzymes

produces active enzymes.

Enzyme synthesis and proteolytic degradation are comparatively slow mechanisms for regulating enzyme concentration, with response times of hours, days or even weeks. Proenzyme activation is a more rapid method of increasing enzyme activity but, as a regulatory mechanism, it has the disadvantage of not

being a reversible process. Proenzymes are generally synthesized in abundance, stored in secretory granules and covalently activated upon release from their

storage sites. Examples of important proenzymes include pepsinogen, trypsinogen and chymotrypsinogen, which give rise to the proteolytic digestive enzymes. Likewise, many of the proteins involved in the cascade of chemical reactions responsible for blood clotting are synthesized as proenzymes. Other

important proteins, such as peptide hormones and collagen, are also derived by covalent modification of precursors.

Another mechanism of regulating enzyme activity is to sequester enzymes in compartments where access to their substrates is limited. For example, the proteolysis of cell proteins and glycolipids by enzymes responsible for their

degradation is controlled by sequestering these enzymes within the lysosome.

In contrast to regulatory mechanisms that alter enzyme concentration, there is an important group of regulatory mechanisms that do not affect enzyme

concentration, are reversible and rapid in action, and actually carry out most of the moment- to- moment physiological regulation of enzyme activity. These mechanisms include allosteric regulation, regulation by reversible covalent

modification and regulation by control proteins such as calmodulin. Reversible covalent modification is a major mechanism for the rapid and transient regulation

of enzyme activity. The best examples, again, come from studies on the regulation of glycogen metabolism where phosphorylation of glycogen synthase

and glycogen phosphorylase kinase results in the stimulation of glycogen degradation while glycogen synthesis is coordinately inhibited. Numerous other

enzymes of intermediary metabolism are affected by phosphorylation, either positively or negatively. These covalent phosphorylations can be reversed by a separate sub-subclass of enzymes known as phosphatases. Recent research has indicated that the aberrant phosphorylation of growth factor and hormone

receptors, as well as of proteins that regulate cell division, often leads to unregulated cell growth or cancer. The usual sites for phosphate addition to proteins are the serine, threonine and tyrosine R group hydroxyl residues.

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Allosteric Enzymes In addition to simple enzymes that interact only with substrates and inhibitors,

there is a class of enzymes that bind small, physiologically important molecules and modulate activity in ways other than those described above. These are

known as allosteric enzymes; the small regulatory molecules to which they bind are known as effectors. Allosteric effectors bring about catalytic modification by binding to the enzyme at distinct allosteric sites, well removed from the catalytic

site, and causing conformational changes that are transmitted through the bulk of the protein to the catalytically active site(s).

The hallmark of effectors is that when they bind to enzymes, they alter the catalytic properties of an enzyme's active site. Those that increase catalytic

activity are known as positive effectors. Effectors that reduce or inhibit catalytic activity are negative effectors.

Most allosteric enzymes are oligomeric (consisting of multiple subunits); generally they are located at or near branch points in metabolic pathways, where they are influential in directing substrates along one or another of the available

metabolic paths. The effectors that modulate the activity of these allosteric enzymes are of two types. Those activating and inhibiting effectors that bind at

allosteric sites are called heterotropic effectors. (Thus there exist both positive and negative heterotropic effectors.) These effectors can assume a vast diversity

of chemical forms, ranging from simple inorganic molecules to complex nucleotides such as cyclic adenosine monophosphate (cAMP). Their single

defining feature is that they are not identical to the substrate. In many cases the substrate itself induces distant allosteric effects when it binds

to the catalytic site. Substrates acting as effectors are said to be homotropic effectors. When the substrate is the effector, it can act as such, either by binding

to the substrate-binding site, or to an allosteric effector site. When the substrate binds to the catalytic site it transmits an activity-modulating effect to other

subunits of the molecule. Often used as the model of a homotropic effector is hemoglobin, although it is not a branch-point enzyme and thus does not fit the

definition on all counts. There are two ways that enzymatic activity can be altered by effectors: the Vmax can be increased or decreased, or the Km can be raised or lowered. Enzymes

whose Km is altered by effectors are said to be K-type enzymes and the effector a K-type effector. If Vmax is altered, the enzyme and effector are said to be V-

type. Many allosteric enzymes respond to multiple effectors with V-type and K-type behavior. Here again, hemoglobin is often used as a model to study

allosteric interactions, although it is not strictly an enzyme. In the preceding discussion we assumed that allosteric sites and catalytic sites were homogeneously present on every subunit of an allosteric enzyme. While

this is often the case, there is another class of allosteric enzymes that are comprised of separate catalytic and regulatory subunits. The archetype of this

class of enzymes is cAMP-dependent protein kinase (PKA), whose mechanism of activation is illustrated. The enzyme is tetrameric, containing two catalytic subunits and two regulatory subunits, and enzymatically inactive. When

intracellular cAMP levels rise, one molecule of cAMP binds to each regulatory subunit, causing the tetramer to dissociate into one regulatory dimer and two catalytic monomers. In the dissociated form, the catalytic subunits are fully

active; they catalyze the phosphorylation of a number of other enzymes, such as those involved in regulating glycogen metabolism. The regulatory subunits have

no catalytic activity. back to the top

Enzymes in the Diagnosis of Pathology

Numerous enzymes have been shown to

LDH occurs in 5 closely related, but slightly different forms (isozymes)

LDH 1 - Found in heart and red-blood cells

LDH 2 - Found in heart and red-blood cells

LDH 3 - Found in a variety of organs

LDH 4 - Found in a variety of organs

LDH 5 - Found in liver and skeletal muscle

CK-1 (BB) is the characteristic isozyme in brain and is in significant amounts in smooth muscle.

CK-3 (MM) is the predominant isozyme in muscle.

CK-2(MB) accounts for about 35% of the CK activity in cardiac muscle, but less than 5% in skeletal muscle.

Since most of the released CK after a myocardial infarction is MM, an increased RATIO of CK-MB to total CK may help in diagnosis of an acute MI, but

an increase of total CK in itself may not.

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Last modified: Wednesday, 27-Aug-2003 10:11:23 EST

Selected by the SciLinks program, a service of National Science Teachers Association. Copyright 2001

WINNER ! !


Introduction to Vitamins Vitamins are organic molecules that function in a wide variety of capacities within the body. The most prominent function is as cofactors for enzymatic reactions. The distinguishing feature of the vitamins is that they generally cannot be synthesized by mammalian cells and, therefore, must be supplied in the diet. The vitamins are of two distinct types:

Water Soluble Vitamins Fat Soluble Vitamins

• Thiamin (B1) • Vitamin A

o B1 Deficiency and Disease

• Riboflavin (B2) o B2 Deficiency and

Disease • Niacin (B3)


• Pantothenic Acid (B5) • Pyridoxal, Pyridoxamine,

Pyridoxine (B6) • Biotin • Cobalamin (B12)


• Folic Acid o Folate Deficiency and

Disease • Ascorbic Acid

o Gene Control by Vitamin A

o Role of Vitamin A in Vision

o Additional Roles of Vitamin A

o Clinical Significances of Vitamin A

• Vitamin D o Clinical Significances of

Vitamin D • Vitamin E

o Clinical Significances of Vitamin E

• Vitamin K o Clinical Significance of

Vitamin K


Thiamin

Thiamin structure

Thiamin is also known as vitamin B1 . Thiamin is derived from a substituted pyrimidine and a thiazole which are coupled by a methylene bridge. Thiamin is rapidly converted to its active form, thiamin pyrophosphate, TPP, in the brain and liver by a specific enzymes, thiamin diphosphotransferase.

Thiamin pyrophosphate

TPP is necessary as a cofactor for the pyruvate and αααα-ketoglutarate dehydrogenase catalyzed reactions as well as the transketolase catalyzed reactions of the pentose phosphate pathway. A deficiency in thiamin intake leads to a severely reduced capacity of cells to generate energy as a result of its role in these reactions. The dietary requirement for thiamin is proportional to the caloric intake of the diet and ranges from 1.0 - 1.5 mg/day for normal adults. If the carbohydrate content of the diet is excessive then an in thiamin intake will be required. back to the top

Clinical Significances of Thiamin Deficiency

The earliest symptoms of thiamin deficiency include constipation, appetite suppression, nausea as well as mental depression, peripheral neuropathy and fatigue. Chronic thiamin deficiency leads to more severe neurological symptoms including ataxia, mental confusion and loss of eye coordination. Other clinical symptoms of prolonged thiamin deficiency are related to cardiovascular and musculature defects. The severe thiamin deficiency disease known as Beriberi, is the result of a diet that is carbohydrate rich and thiamin deficient. An additional thiamin deficiency related disease is known as Wernicke-Korsakoff syndrome. This disease is most commonly found in chronic alcoholics due to their poor dietetic lifestyles. back to the top

Riboflavin

Riboflavin structure

Riboflavin is also known as vitamin B2. Riboflavin is the precursor for the coenzymes, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). The enzymes that require FMN or FAD as cofactors are termed flavoproteins. Several flavoproteins also contain metal ions and are termed metalloflavoproteins. Both classes of enzymes are involved in a wide range of redox reactions, e.g. succinate dehydrogenase and xanthine oxidase. During the course of the enzymatic reactions involving the flavoproteins the reduced forms of FMN and FAD are formed, FMNH2 and FADH2, respectively.

Structure of FAD nitrogens 1 & 5 carry hydrogens in FADH2

The normal daily requirement for riboflavin is 1.2 - 1.7 mg/day for normal adults. back to the top

Clinical Significances of Flavin Deficiency

Riboflavin deficiencies are rare in the United States due to the presence of adequate amounts of the vitamin in eggs, milk, meat and cereals. Riboflavin deficiency is often seen in chronic alcoholics due to their poor dietetic habits. Symptoms associated with riboflavin deficiency include, glossitis, seborrhea, angular stomatitis, cheilosis and photophobia. Riboflavin decomposes when exposed to visible light. This characteristic can lead to riboflavin deficiencies in newborns treated for hyperbilirubinemia by phototherapy. back to the top

Niacin

Nicotinamide Nicotinic Acid

Niacin (nicotinic acid and nicotinamide) is also known as vitamin B3. Both nicotinic acid and nicotinamide can serve as the dietary source of vitamin B3. Niacin is required for the synthesis of the active forms of vitamin B3, nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+). Both NAD+ and NADP+ function as cofactors for numerous dehydrogenase, e.g., lactate and malate dehydrogenases.

Structure of NAD+ NADH is shown in the box insert.

The -OH phosphorylated in NADP+ is indicated by the red arrow.

Niacin is not a true vitamin in the strictest definition since it can be derived from the amino acid tryptophan. However, the ability to utilize tryptophan for niacin synthesis is inefficient (60 mg of tryptophan are required to synthesize 1 mg of

niacin). Also, synthesis of niacin from tryptophan requires vitamins B1, B2 and B6 which would be limiting in themselves on a marginal diet. The recommended daily requirement for niacin is 13 - 19 niacin equivalents (NE) per day for a normal adult. One NE is equivalent to 1 mg of free niacin). back to the top

Clinical Significances of Niacin and Nicotinic Acid A diet deficient in niacin (as well as tryptophan) leads to glossitis of the tongue, dermatitis, weight loss, diarrhea, depression and dementia. The severe symptoms, depression, dermatitis and diarrhea, are associated with the condition known as pellagra. Several physiological conditions (e.g. Hartnup disease and malignant carcinoid syndrome) as well as certain drug therapies (e.g. isoniazid) can lead to niacin deficiency. In Hartnup disease tryptophan absorption is impaired and in malignant carcinoid syndrome tryptophan metabolism is altered resulting in excess serotonin synthesis. Isoniazid (the hydrazide derivative of isonicotinic acid) is the primary drug for chemotherapy of tuberculosis. Nicotinic acid (but not nicotinamide) when administered in pharmacological doses of 2 - 4 g/day lowers plasma cholesterol levels and has been shown to be a useful therapeutic for hypercholesterolemia. The major action of nicotinic acid in this capacity is a reduction in fatty acid mobilization from adipose tissue. Although nicotinic acid therapy lowers blood cholesterol it also causes a depletion of glycogen stores and fat reserves in skeletal and cardiac muscle. Additionally, there is an elevation in blood glucose and uric acid production. For these reasons nicotinic acid therapy is not recommended for diabetics or persons who suffer from gout. back to the top

Pantothenic Acid

Pantothenic Acid

Pantothenic acid is also known as vitamin B5. Pantothenic acid is formed from β-alanine and pantoic acid. Pantothenate is required for synthesis of coenzyme A, CoA and is a component of the acyl carrier protein (ACP) domain of fatty acid synthase. Pantothenate is, therefore, required for the metabolism of carbohydrate via the TCA cycle and all fats and proteins. At least 70 enzymes have been identified as requiring CoA or ACP derivatives for their function.

Deficiency of pantothenic acid is extremely rare due to its widespread distribution in whole grain cereals, legumes and meat. Symptoms of pantothenate deficiency are difficult to assess since they are subtle and resemble those of other B vitamin deficiencies.

Coenzyme A

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Vitamin B6

Pyridoxine Pyridoxal Pyridoxamine

Pyridoxal, pyridoxamine and pyridoxine are collectively known as vitamin B6. All three compounds are efficiently converted to the biologically active form of vitamin B6, pyridoxal phosphate. This conversion is catalyzed by the ATP requiring enzyme, pyridoxal kinase.

Pyridoxal Phosphate

Pyridoxal phosphate functions as a cofactor in enzymes involved in transamination reactions required for the synthesis and catabolism of the amino acids as well as in glycogenolysis as a cofactor for glycogen phosphorylase. The requirement for vitamin B6 in the diet is proportional to the level of protein consumption ranging from 1.4 - 2.0 mg/day for a normal adult. During pregnancy and lactation the requirement for vitamin B6 increases approximately 0.6 mg/day. Deficiencies of vitamin B6 are rare and usually are related to an overall deficiency of all the B-complex vitamins. Isoniazid (see niacin deficiencies above) and penicillamine (used to treat rheumatoid arthritis and cystinurias) are two drugs that complex with pyridoxal and pyridoxal phosphate resulting in a deficiency in this vitamin. back to the top

Biotin

Biotin

Biotin is the cofactor required of enzymes that are involved in carboxylation reactions, e.g. acetyl-CoA carboxylase and pyruvate carboxylase. Biotin is found in numerous foods and also is synthesized by intestinal bacteria and as such deficiencies of the vitamin are rare. Deficiencies are generally seen only after long antibiotic therapies which deplete the intestinal fauna or following excessive consumption of raw eggs. The latter is due to the affinity of the egg white protein, avidin, for biotin preventing intestinal absorption of the biotin. back to the top

Cobalamin

Cobalamin is more commonly known as vitamin B12. Vitamin B12 is composed of a complex tetrapyrrol ring structure (corrin ring) and a cobalt ion in the center. Vitamin B12 is synthesized exclusively by microorganisms and is found in the liver of animals bound to protein as methycobalamin or 5'-deoxyadenosylcobalamin. The vitamin must be hydrolyzed from protein in order to be active. Hydrolysis occurs in the stomach by gastric acids or the intestines by trypsin digestion following consumption of animal meat. The vitamin is then bound by intrinsic factor, a protein secreted by parietal cells of the stomach, and carried to the

ileum where it is absorbed. Following absorption the vitamin is transported to the liver in the blood bound to transcobalamin II. There are only two clinically significant reactions in the body that require vitamin B12 as a cofactor. During the catabolism of fatty acids with an odd number of carbon atoms and the amino acids valine, isoleucine and threonine the resultant propionyl-CoA is converted to succinyl-CoA for oxidation in the TCA cycle. One of the enzymes in this pathway, methylmalonyl-CoA mutase, requires vitamin B12 as a cofactor in the conversion of methylmalonyl-CoA to succinyl-CoA. The 5'-deoxyadenosine derivative of cobalamin is required for this reaction. The second reaction requiring vitamin B12 catalyzes the conversion of homocysteine to methionine and is catalyzed by methionine synthase. This reaction results in the transfer of the methyl group from N5-methyltetrahydrofolate to hydroxycobalamin generating tetrahydrofolate (THF) and methylcobalamin during the process of the conversion.

Deoxyadenosylcobalamin

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Clinical Significances of B12 Deficiency

The liver can store up to six years worth of vitamin B12, hence deficiencies in this vitamin are rare. Pernicious anemia is a megaloblastic anemia resulting from vitamin B12 deficiency that develops as a result a lack of intrinsic factor in the stomach leading to malabsorption of the vitamin. The anemia results from impaired DNA synthesis due to a block in purine and thymidine biosynthesis. The block in nucleotide biosynthesis is a consequence of the effect of vitamin B12 on folate metabolism. When vitamin B12 is deficient essentially all of the folate becomes trapped as the N5-methylTHF derivative as a result of the loss of functional methionine synthase. This trapping prevents the synthesis of other THF derivatives required for the purine and thymidine nucleotide biosynthesis pathways. Neurological complications also are associated with vitamin B12 deficiency and result from a progressive demyelination of nerve cells. The demyelination is thought to result from the increase in methylmalonyl-CoA that result from vitamin B12 deficiency. Methylmalonyl-CoA is a competitive inhibitor of malonyl-CoA in fatty acid biosynthesis as well as being able to substitute for malonyl-CoA in any fatty acid biosynthesis that may occur. Since the myelin sheath is in continual flux the methylmalonyl-CoA-induced inhibition of fatty acid synthesis results in the eventual destruction of the sheath. The incorporation methylmalonyl-CoA into fatty acid biosynthesis results in branched-chain fatty acids being produced that may severely alter the architecture of the normal membrane structure of nerve cells back to the top

Folic Acid

Folic Acid positions 7 & 8 carry hydrogens in dihydrofolate (DHF) positions 5-8 carry hydrogens in tetrahydrofolate (THF)

Folic acid is a conjugated molecule consisting of a pteridine ring structure linked to para-aminobenzoic acid (PABA) that forms pteroic acid. Folic acid itself is then generated through the conjugation of glutamic acid residues to pteroic acid. Folic acid is obtained primarily from yeasts and leafy vegetables as well as animal liver. Animal cannot synthesize PABA nor attach glutamate residues to pteroic acid, thus, requiring folate intake in the diet. When stored in the liver or ingested folic acid exists in a polyglutamate form. Intestinal mucosal cells remove some of the glutamate residues through the

action of the lysosomal enzyme, conjugase. The removal of glutamate residues makes folate less negatively charged (from the polyglutamic acids) and therefore more capable of passing through the basal lamenal membrane of the epithelial cells of the intestine and into the bloodstream. Folic acid is reduced within cells (principally the liver where it is stored) to tetrahydrofolate (THF also H4folate) through the action of dihydrofolate reductase (DHFR), an NADPH-requiring enzyme. The function of THF derivatives is to carry and transfer various forms of one carbon units during biosynthetic reactions. The one carbon units are either methyl, methylene, methenyl, formyl or formimino groups.

Active center of tetrahydrofolate (THF). Note that the N5 position is the site of attachment of methyl groups, the N10 the site for attachment of formyl and formimino groups and that both N5 and N10 bridge the methylene and methenyl groups.

These one carbon transfer reactions are required in the biosynthesis of serine, methionine, glycine, choline and the purine nucleotides and dTMP.

The ability to acquire choline and amino acids from the diet and to salvage the purine nucleotides makes the role of N5,N10-methylene-THF in dTMP synthesis

the most metabolically significant function for this vitamin. The role of vitamin B12 and N5-methyl-THF in the conversion of homocysteine to methionine also can

have a significant impact on the ability of cells to regenerate needed THF. back to the top

Clinical Significance of Folate Deficiency

Folate deficiency results in complications nearly identical to those described for vitamin B12 deficiency. The most pronounced effect of folate deficiency on cellular

processes is upon DNA synthesis. This is due to an impairment in dTMP synthesis which leads to cell cycle arrest in S-phase of rapidly proliferating cells,

in particular hematopoietic cells. The result is megaloblastic anemia as for vitamin B12 deficiency. The inability to synthesize DNA during erythrocyte

maturation leads to abnormally large erythrocytes termed macrocytic anemia. Folate deficiencies are rare due to the adequate presence of folate in food. Poor

dietary habits as those of chronic alcoholics can lead to folate deficiency. The predominant causes of folate deficiency in non-alcoholics are impaired absorption or metabolism or an increased demand for the vitamin. The

predominant condition requiring an increase in the daily intake of folate is pregnancy. This is due to an increased number of rapidly proliferating cells

present in the blood. The need for folate will nearly double by the third trimester of pregnancy. Certain drugs such as anticonvulsants and oral contraceptives can

impair the absorption of folate. Anticonvulsants also increase the rate of folate metabolism.

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Ascorbic Acid

Ascorbic Acid

Ascorbic acid is more commonly known as vitamin C. Ascorbic acid is derived

from glucose via the uronic acid pathway. The enzyme L-gulonolactone oxidase responsible for the conversion of gulonolactone to ascorbic acid is

absent in primates making ascorbic acid required in the diet. The active form of vitamin C is ascorbate acid itself. The main function of

ascorbate is as a reducing agent in a number of different reactions. Vitamin C has the potential to reduce cytochromes a and c of the respiratory chain as well

as molecular oxygen. The most important reaction requiring ascorbate as a cofactor is the hydroxylation of proline residues in collagen. Vitamin C is,

therefore, required for the maintenance of normal connective tissue as well as for wound healing since synthesis of connective tissue is the first event in wound tissue remodeling. Vitamin C also is necessary for bone remodeling due to the

presence of collagen in the organic matrix of bones. Several other metabolic reactions require vitamin C as a cofactor. These include the catabolism of tyrosine and the synthesis of epinephrine from tyrosine and the

synthesis of the bile acids. It is also believed that vitamin C is involved in the

process of steroidogenesis since the adrenal cortex contains high levels of vitamin C which are depleted upon adrenocorticotropic hormone (ACTH)

stimulation of the gland. Deficiency in vitamin C leads to the disease scurvy due to the role of the vitamin

in the post-translational modification of collagens. Scurvy is characterized by easily bruised skin, muscle fatigue, soft swollen gums, decreased wound healing and hemorrhaging, osteoporosis, and anemia. Vitamin C is readily absorbed and

so the primary cause of vitamin C deficiency is poor diet and/or an increased requirement. The primary physiological state leading to an increased requirement for vitamin C is severe stress (or trauma). This is due to a rapid depletion in the adrenal stores of the vitamin. The reason for the decrease in adrenal vitamin C levels is unclear but may be due either to redistribution of the vitamin to areas

that need it or an overall increased utilization. back to the top

Vitamin A

Vitamin A consists of three biologically active molecules, retinol, retinal (retinaldehyde) and retinoic acid.

All-trans-retinal 11-cis-retinal

Retinol Retinoic Acid

Each of these compounds are derived from the plant precursor molecule, ββββ-carotene (a member of a family of molecules known as carotenoids). Beta-carotene, which consists of two molecules of retinal linked at their aldehyde

ends, is also referred to as the provitamin form of vitamin A.

Ingested β-carotene is cleaved in the lumen of the intestine by ββββ-carotene dioxygenase to yield retinal. Retinal is reduced to retinol by retinaldehyde

reductase, an NADPH requiring enzyme within the intestines. Retinol is esterified to palmitic acid and delivered to the blood via chylomicrons. The uptake of chylomicron remnants by the liver results in delivery of retinol to this organ for

storage as a lipid ester within lipocytes. Transport of retinol from the liver to extrahepatic tissues occurs by binding of hydrolyzed retinol to aporetinol

binding protein (RBP). the retinol-RBP complex is then transported to the cell surface within the Golgi and secreted. Within extrahepatic tissues retinol is bound to cellular retinol binding protein (CRBP). Plasma transport of retinoic acid is

accomplished by binding to albumin. back to the top

Gene Control Exerted by Retinol and Retinoic Acid

Within cells both retinol and retinoic acid bind to specific receptor proteins. Following binding, the receptor-vitamin complex interacts with specific sequences in several genes involved in growth and differentiation and affects expression of

these genes. In this capacity retinol and retinoic acid are considered hormones of the steroid/thyroid hormone superfamily of proteins. Vitamin D also acts in a similar capacity. Several genes whose patterns of expression are altered by

retinoic acid are involved in the earliest processes of embryogenesis including the differentiation of the three germ layers, organogenesis and limb development.

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Vision and the Role of Vitamin A Photoreception in the eye is the function of two specialized cell types located in

the retina; the rod and cone cells. Both rod and cone cells contain a photoreceptor pigment in their membranes. The photosensitive compound of

most mammalian eyes is a protein called opsin to which is covalently coupled an aldehyde of vitamin A. The opsin of rod cells is called scotopsin. The

photoreceptor of rod cells is specifically called rhodopsin or visual purple. This compound is a complex between scotopsin and the 11-cis-retinal (also called 11-cis-retinene) form of vitamin A. Rhodopsin is a serpentine receptor imbedded in the membrane of the rod cell. Coupling of 11-cis-retinal occurs at three of the

transmembrane domains of rhodopsin. Intracellularly, rhodopsin is coupled to a specific G-protein called transducin.

When the rhodopsin is exposed to light it is bleached releasing the 11-cis-retinal from opsin. Absorption of photons by 11-cis-retinal triggers a series of

conformational changes on the way to conversion all-trans-retinal. One important conformational intermediate is metarhodopsin II. The release of opsin

results in a conformational change in the photoreceptor. This conformational change activates transducin, leading to an increased GTP-binding by the a-

subunit of transducin. Binding of GTP releases the α-subunit from the inhibitory β- and γ-subunits. The GTP-activated α-subunit in turn activates an associated phosphodiesterase; an enzyme that hydrolyzes cyclic-GMP (cGMP) to GMP. Cyclic GMP is required to maintain the Na+ channels of the rod cell in the open

conformation. The drop in cGMP concentration results in complete closure of the Na+ channels. Metarhodopsin II appears to be responsible for initiating the

closure of the channels. The closing of the channels leads to hyperpolarization of the rod cell with concomitant propagation of nerve impulses to the brain.

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Additional Role of Retinol Retinol also functions in the synthesis of certain glycoproteins and

mucopolysaccharides necessary for mucous production and normal growth regulation. This is accomplished by phosphorylation of retinol to retinyl

phosphate which then functions similarly to dolichol phosphate. back top the top

Clinical Significances of Vitamin A Deficiency

Vitamin A is stored in the liver and deficiency of the vitamin occurs only after prolonged lack of dietary intake. The earliest symptoms of vitamin A deficiency

are night blindness. Additional early symptoms include follicular hyperkeratinosis, increased susceptibility to infection and cancer and anemia

equivalent to iron deficient anemia. Prolonged lack of vitamin A leads to deterioration of the eye tissue through progressive keratinization of the cornea, a

condition known as xerophthalmia. The increased risk of cancer in vitamin deficiency is thought to be the result of a

depletion in β-carotene. Beta-carotene is a very effective antioxidant and is suspected to reduce the risk of cancers known to be initiated by the production of free radicals. Of particular interest is the potential benefit of increased β-carotene intake to reduce the risk of lung cancer in smokers. However, caution needs to be taken when increasing the intake of any of the lipid soluble vitamins. Excess

accumulation of vitamin A in the liver can lead to toxicity which manifests as bone pain, hepatosplenomegaly, nausea and diarrhea.

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Vitamin D Vitamin D is a steroid hormone that functions to regulate specific gene

expression following interaction with its intracellular receptor. The biologically active form of the hormone is 1,25-dihydroxy vitamin D3 (1,25-(OH)2D3, also

termed calcitriol). Calcitriol functions primarily to regulate calcium and phosphorous homeostasis.

Ergosterol Vitamin D2

7-Dehydrocholesterol Vitamin D3

Active calcitriol is derived from ergosterol (produced in plants) and from 7-dehydrocholesterol (produced in the skin). Ergocalciferol (vitamin D2) is formed by uv irradiation of ergosterol. In the skin 7-dehydrocholesterol is

converted to cholecalciferol (vitamin D3) following uv irradiation. Vitamin D2 and D3 are processed to D2-calcitriol and D3-calcitriol, respectively, by the same enzymatic pathways in the body. Cholecalciferol (or egrocalciferol) are

absorbed from the intestine and transported to the liver bound to a specific vitamin D-binding protein. In the liver cholecalciferol is hydroxylated at the 25 position by a specific D3-25-hydroxylase generating 25-hydroxy-D3 [25-(OH)D3] which is the major circulating form of vitamin D. Conversion of 25-(OH)D3 to its biologically active form, calcitriol, occurs through the activity of a specific D3-1-hydroxylase present in the proximal convoluted tubules of the kidneys, and in bone and placenta. 25-(OH)D3 can also be hydroxylated at the 24 position by a

specific D3-24-hydroxylase in the kidneys, intestine, placenta and cartilage.

25-hydroxyvitamin D3 1,25-dihydroxyvitamin D3

Calcitriol functions in concert with parathyroid hormone (PTH) and calcitonin to regulate serum calcium and phosphorous levels. PTH is released in response

to low serum calcium and induces the production of calcitriol. In contrast, reduced levels of PTH stimulate synthesis of the inactive 24,25-(OH)2D3. In the

intestinal epithelium, calcitriol functions as a steroid hormone in inducing the expression of calbindinD28K, a protein involved in intestinal calcium absorption. The increased absorption of calcium ions requires concomitant absorption of a

negatively charged counter ion to maintain electrical neutrality. The predominant counter ion is Pi. When plasma calcium levels fall the major sites of action of

calcitriol and PTH are bone where they stimulate bone resorption and the kidneys where they inhibit calcium excretion by stimulating reabsorption by the

distal tubules. The role of calcitonin in calcium homeostasis is to decrease elevated serum calcium levels by inhibiting bone resorption.

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Clinical Significance of Vitamin D Deficiency As a result of the addition of vitamin D to milk, deficiencies in this vitamin are rare in this country. The main symptom of vitamin D deficiency in children is rickets and in adults is osteomalacia. Rickets is characterized improper mineralization

during the development of the bones resulting in soft bones. Osteomalacia is characterized by demineralization of previously formed bone leading to increased

softness and susceptibility to fracture. back to the top

Vitamin E

αααα-Tocopherol

Vitamin E is a mixture of several related compounds known as tocopherols. The

α-tocopherol molecule is the most potent of the tocopherols. Vitamin E is absorbed from the intestines packaged in chylomicrons. It is delivered to the tissues via chylomicron transport and then to the liver through chylomicron

remnant uptake. The liver can export vitamin E in VLDLs. Due to its lipophilic nature, vitamin E accumulates in cellular membranes, fat deposits and other

circulating lipoproteins. The major site of vitamin E storage is in adipose tissue. The major function of vitamin E is to act as a natural antioxidant by scavenging

free radicals and molecular oxygen. In particular vitamin E is important for preventing peroxidation of polyunsaturated membrane fatty acids. The vitamins E and C are interrelated in their antioxidant capabilities. Active α-tocopherol can be

regenerated by interaction with vitamin C following scavenge of a peroxy free radical. Alternatively, α-tocopherol can scavenge two peroxy free radicals and

then be conjugated to glucuronate for excretion in the bile. back to the top

Clinical significances of Vitamin E Deficiency No major disease states have been found to be associated with vitamin E deficiency due to adequate levels in the average American diet. The major symptom of vitamin E deficiency in humans is an increase in red blood cell

fragility. Since vitamin E is absorbed from the intestines in chylomicrons, any fat malabsorption diseases can lead to deficiencies in vitamin E intake. Neurological disorders have been associated with vitamin E deficiencies associated with fat

malabsorptive disorders. Increased intake of vitamin E is recommended in premature infants fed formulas that are low in the vitamin as well as in persons

consuming a diet high in polyunsaturated fatty acids. Polyunsaturated fatty acids tend to form free radicals upon exposure to oxygen and this may lead to an

increased risk of certain cancers. back to the top

Vitamin K

The K vitamins exist naturally as K1 (phylloquinone) in green vegetables and K2 (menaquinone) produced by intestinal bacteria and K3 is synthetic menadione.

When administered, vitamin K3 is alkylated to one of the vitamin K2 forms of menaquinone.

Vitamin K1 Vitamin K2

"n" can be 6, 7 or 9 isoprenoid groups

Vitamin K3

The major function of the K vitamins is in the maintenance of normal levels of the blood clotting proteins, factors II, VII, IX, X and protein C and protein S, which

are synthesized in the liver as inactive precursor proteins. Conversion from inactive to active clotting factor requires a posttranslational modification of

specific glutamate (E) residues. This modification is a carboxylation and the enzyme responsible requires vitamin K as a cofactor. The resultant modified E

residues are γγγγ-carboxyglutamate (gla). This process is most clearly understood for factor II, also called preprothrombin. Prothrombin is modified

preprothrombin. The gla residues are effective calcium ion chelators. Upon chelation of calcium, prothrombin interacts with phospholipids in membranes and

is proteolysed to thrombin through the action of activated factor X (Xa). During the carboxylation reaction reduced hydroquinone form of vitamin K is converted to a 2,3-epoxide form. The regeneration of the hydroquinone form

requires an uncharacterized reductase. This latter reaction is the site of action of the dicumarol based anticoagulants such as warfarin.

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Clinical significance of Vitamin K Deficiency Naturally occurring vitamin K is absorbed from the intestines only in the presence of bile salts and other lipids through interaction with chylomicrons. Therefore, fat malabsorptive diseases can result in vitamin K deficiency. The synthetic vitamin K3 is water soluble and absorbed irrespective of the presence of intestinal lipids

and bile. Since the vitamin K2 form is synthesized by intestinal bacteria, deficiency of the vitamin in adults is rare. However, long term antibiotic treatment

can lead to deficiency in adults. The intestine of newborn infants is sterile, therefore, vitamin K deficiency in infants is possible if lacking from the early diet.

The primary symptom of a deficiency in infants is a hemorrhagic syndrome. back to the top




• Digestion of Dietary Carbohydrates • The Energy Derived from Glycolysis • Reactions of Glycolysis • Images of the Pathway of Glycolysis • Anaerobic Glycolysis • Regulation of Glycolysis • Metabolic Fates of Pyruvate • Lactate Metabolism • Ethanol Metabolism • Entry of Non-Glucose Carbons into Glycolysis • Glycogen Metabolism • Regulation of Blood Glucose Levels


Digestion of Dietary Carbohydrates Dietary carbohydrate from which humans gain energy enter the body in complex forms, such as disaccharides and the polymers starch (amylose and amylopectin) and glycogen. The polymer cellulose is also consumed but not digested. The first step in the metabolism of digestible carbohydrate is the conversion of the higher polymers to simpler, soluble forms that can be transported across the intestinal wall and delivered to the tissues. The breakdown of polymeric sugars begins in the mouth. Saliva has a slightly acidic pH of 6.8 and contains lingual amylase that begins the digestion of carbohydrates. The action of lingual amylase is limited to the area of the mouth and the esophagus; it is virtually inactivated by the much stronger acid pH of the stomach. Once the food has arrived in the stomach, acid hydrolysis contributes to its degradation; specific gastric proteases and lipases aid this process for proteins and fats, respectively. The mixture of gastric secretions, saliva, and food, known collectively as chyme, moves to the small intestine. The main polymeric-carbohydrate digesting enzyme of the small intestine is αααα-amylase. This enzyme is secreted by the pancreas and has the same activity as salivary amylase, producing disaccharides and trisaccharides. The latter are converted to monosaccharides by intestinal saccharidases, including maltases

that hydrolyze di- and trisaccharides, and the more specific disaccharidases, sucrase, lactase, and trehalase. The net result is the almost complete conversion of digestible carbohydrate to its constituent monosaccharides. The resultant glucose and other simple carbohydrates are transported across the intestinal wall to the hepatic portal vein and then to liver parenchymal cells and other tissues. There they are converted to fatty acids, amino acids, and glycogen, or else oxidized by the various catabolic pathways of cells. Oxidation of glucose is known as glycolysis.Glucose is oxidized to either lactate or pyruvate. Under aerobic conditions, the dominant product in most tissues is pyruvate and the pathway is known as aerobic glycolysis. When oxygen is depleted, as for instance during prolonged vigorous exercise, the dominant glycolytic product in many tissues is lactate and the process is known as anaerobic glycolysis. back to the top

The Energy Derived from Glucose Oxidation

Aerobic glycolysis of glucose to pyruvate, requires two equivalents of ATP to activate the process, with the subsequent production of four equivalents of ATP and two equivalents of NADH. Thus, conversion of one mole of glucose to two moles of pyruvate is accompanied by the net production of two moles each of ATP and NADH.

Glucose + 2 ADP + 2 NAD+ + 2 Pi -----> 2 Pyruvate + 2 ATP + 2 NADH + 2 H+

The NADH generated during glycolysis is used to fuel mitochondrial ATP synthesis via oxidative phosphorylation, producing either two or three equivalents of ATP depending upon whether the glycerol phosphate shuttle or the malate-aspartate shuttle is used to transport the electrons from cytoplasmic NADH into the mitochondria. The net yield from the oxidation of 1 mole of glucose to 2 moles of pyruvate is, therefore, either 6 or 8 moles of ATP. Complete oxidation of the 2 moles of pyruvate, through the TCA cycle, yeilds an additional 30 moles of ATP; the total yield, therefore being either 36 or 38 moles of ATP from the complete oxidation of 1 mole of glucose to CO2 and H2O. back to the top

The Individual Reactions of Glycolysis

The pathway of glycolysis can be seen as consisting of 2 separate phases. The first is the chemical priming phase requiring energy in the form of ATP, and the second is considered the energy-yielding phase. In the first phase, 2 equivalents of ATP are used to convert glucose to fructose 1,6-bisphosphate (F1,6BP). In the second phase F1,6BP is degraded to pyruvate, with the production of 4 equivalents of ATP and 2 equivalents of NADH.

Pathway of glycolysis from glucose to pyruvate. Substrates and products are in blue, enzymes are in green. The two high energy intermediates whose oxidations are coupled to ATP synthesis are shown in red (1,3-bisphosphoglycerate and phosphoenolpyruvate).

The Hexokinase Reaction:

The ATP-dependent phosphorylation of glucose to form glucose 6-phosphate (G6P)is the first reaction of glycolysis, and is catalyzed by tissue-specific isoenzymes known as hexokinases. The phosphorylation accomplishes two goals: First, the hexokinase reaction converts nonionic glucose into an anion that is trapped in the cell, since cells lack transport systems for phosphorylated sugars. Second, the otherwise biologically inert glucose becomes activated into a labile form capable of being further metabolized.

Four mammalian isozymes of hexokinase are known (Types I - IV), with the Type IV isozyme often referred to as glucokinase. Glucokinase is the form of the enzyme found in hepatocytes. The high Km of glucokinase for glucose means that this enzyme is saturated only at very high concentrations of substrate.

Comparison of the activities of hexokinase and glucokinase. The Km for hexokinase is significantly lower (0.1mM) than that of glucokinase (10mM). This difference ensures that non-hepatic tissues (which contain hexokinase) rapidly and efficiently trap blood glucose within their cells by converting it to glucose-6-phosphate. One major function of the liver is to deliver glucose to the blood and this in ensured by having a glucose phosphorylating enzyme (glucokinase) whose Km for glucose is sufficiently higher that the normal circulating concentration of glucose (5mM).

This feature of hepatic glucokinase allows the liver to buffer blood glucose. After meals, when postprandial blood glucose levels are high, liver glucokinase is significantly active, which causes the liver preferentially to trap and to store circulating glucose. When blood glucose falls to very low levels, tissues such as liver and kidney---which contain glucokinases but are not highly dependent on glucose---do not continue to use the meager glucose supplies that remain available. At the same time, tissues such as the brain, which are critically dependent on glucose, continue to scavenge blood glucose using their low Km hexokinases, and as a consequence their viability is protected. Under various

conditions of glucose deficiency, such as long periods between meals, the liver is stimulated to supply the blood with glucose through the pathway of gluconeogenesis. The levels of glucose produced during gluconeogenesis are insufficient to activate glucokinase, allowing the glucose to pass out of hepatocytes and into the blood. The regulation of hexokinase and glucokinase activities is also different. Hexokinases I, II, and III are allosterically inhibited by product (G6P) accumulation, whereas glucokinases are not. The latter further insures liver accumulation of glucose stores during times of glucose excess, while favoring peripheral glucose utilization when glucose is required to supply energy to peripheral tissues.

Phosphohexose Isomerase:

The second reaction of glycolysis is an isomerization, in which G6P is converted to fructose 6-phosphate (F6P). The enzyme catalyzing this reaction is phosphohexose isomerase (also known as phosphoglucose isomerase). The reaction is freely reversible at normal cellular concentrations of the two hexose phosphates and thus catalyzes this interconversion during glycolytic carbon flow and during gluconeogenesis.

6-Phosphofructo-1-Kinase (Phosphofructokinase-1, PFK-1):

The next reaction of glycolysis involves the utilization of a second ATP to convert F6P to fructose 1,6-bisphosphate (F1,6BP). This reaction is catalyzed by 6-phosphofructo-1-kinase, better known as phosphofructokinase-1 or PFK-1. This reaction is not readily reversible because of its large positive free energy (∆G0' = +5.4 kcal/mol) in the reverse direction. Nevertheless, fructose units readily flow in the reverse (gluconeogenic) direction because of the ubiquitous presence of the hydrolytic enzyme, fructose-1,6-bisphosphatase (F-1,6-BPase). The presence of these two enzymes in the same cell compartment provides an example of a metabolic futile cycle, which if unregulated would rapidly deplete cell energy stores. However, the activity of these two enzymes is so highly regulated that PFK-1 is considered to be the rate-limiting enzyme of glycolysis and F-1,6-BPase is considered to be the rate-limiting enzyme in gluconeogenesis.

Aldolase:

Aldolase catalyses the hydrolysis of F1,6BP into two 3-carbon products: dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (G3P).

The aldolase reaction proceeds readily in the reverse direction, being utilized for both glycolysis and gluconeogenesis.

Triose Phosphate Isomerase: The two products of the aldolase reaction equilibrate readily in a reaction catalyzed by triose phosphate isomerase. Succeeding reactions of glycolysis utilize G3P as a substrate; thus, the aldolase reaction is pulled in the glycolytic direction by mass action principals.

Glyceraldehyde-3-Phosphate Dehydrogenase:

The second phase of glucose catabolism features the energy-yielding glycolytic reactions that produce ATP and NADH. In the first of these reactions, glyceraldehyde-3-P dehydrogenase (G3PDH) catalyzes the NAD+-dependent oxidation of G3P to 1,3-bisphosphoglycerate (1,3BPG) and NADH. The G3PDH reaction is reversible, and the same enzyme catalyzes the reverse reaction during gluconeogenesis.

Phosphoglycerate Kinase:

The high-energy phosphate of 1,3-BPG is used to form ATP and 3-phosphoglycerate (3PG) by the enzyme phosphoglycerate kinase. Note that this is the only reaction of glycolysis or gluconeogenesis that involves ATP and yet is reversible under normal cell conditions. Associated with the phosphoglycerate kinase pathway is an important reaction of erythrocytes, the formation of 2,3BPG by the enzyme bisphosphoglycerate mutase. 2,3BPG is an important regulator of hemoglobin's affinity for oxygen. Note that 2,3-bisphosphoglycerate phosphatase degrades 2,3BPG to 3-phosphoglycerate, a normal intermediate of glycolysis. The 2,3BPG shunt thus operates with the expenditure of 1 equivalent of ATP per triose passed through the shunt. The process is not reversible under physiological conditions.

Phosphoglycerate Mutase and Enolase:

The remaining reactions of glycolysis are aimed at converting the relatively low energy phosphoacyl-ester of 3PG to a high-energy form and harvesting the phosphate as ATP. The 3PG is first converted to 2PG by phosphoglycerate mutase and the 2PG conversion to phosphoenoylpyruvate (PEP) is catalyzed by enolase

Pyruvate Kinase:

The final reaction of aerobic glycolysis is catalyzed by the highly regulated enzyme pyruvate kinase (PK). In this strongly exergonic reaction, the high-

energy phosphate of PEP is conserved as ATP. The loss of phosphate by PEP leads to the production of pyruvate in an unstable enol form, which spontaneously tautomerizes to the more stable, keto form of pyruvate. This reaction contributes a large proportion of the free energy of hydrolysis of PEP. back to the top

Anaerobic Glycolysis

Under aerobic conditions, pyruvate in most cells is further metabolized via the TCA cycle. Under anaerobic conditions and in erythrocytes under aerobic conditions, pyruvate is converted to lactate by the enzyme lactate dehydrogenase (LDH), and the lactate is transported out of the cell into the circulation. The conversion of pyruvate to lactate, under anaerobic conditions, provides the cell with a mechanism for the oxidation of NADH (produced during the G3PDH reaction) to NAD+; which occurs during the LDH catalyzed reaction. This reduction is required since NAD+ is a necessary substrate for G3PDH, without which glycolysis will cease. Normally, during aerobic glycolysis the electrons of cytoplasmic NADH are transferred to mitochondrial carriers of the oxidative phosphorylation pathway generating a continuous pool of cytoplasmic NAD+. Aerobic glycolysis generates substantially more ATP per mole of glucose oxidized than does anaerobic glycolysis. The utility of anaerobic glycolysis to a muscle cell when it needs large amounts of energy stems from the fact that the rate of ATP production from glycolysis is approximately 100X faster than from oxidative phosphorylation. During exertion muscle cells do not need to energize anabolic reaction pathways. The requirement is to generate the maximum amount of ATP, for muscle contraction, in the shortest time frame. This is why muscle cells derive almost all of the ATP consumed during exertion from anaerobic glycolysis. back to the top

Regulation of Glycolysis

The reactions catalyzed by hexokinase, PFK-1 and PK all proceed with a relatively large free energy decrease. These nonequilibrium reactions of glycolysis would be ideal candidates for regulation of the flux through glycolysis. Indeed, in vitro studies have shown all three enzymes to be allosterically controlled. Regulation of hexokinase, however, is not the major control point in glycolysis. This is due to the fact that large amounts of G6P are derived from the breakdown of glycogen (the predominant mechanism of carbohydrate entry into glycolysis in skeletal muscle) and, therefore, the hexokinase reaction is not necessary. Regulation of PK is important for reversing glycolysis when ATP is high in order to activate gluconeogenesis. As such this enzyme catalyzed reaction is not a major control point in glycolysis. The rate limiting step in glycolysis is the reaction catalyzed by PFK-1.

PFK-1 is a tetrameric enzyme that exist in two conformational states termed R and T that are in equilibrium. ATP is both a substrate and an allosteric inhibitor of PFK-1. Each subunit has two ATP binding sites, a substrate site and an inhibitor site. The substrate site binds ATP equally well when the tetramer is in either conformation. The inhibitor site binds ATP essentially only when the enzyme is in the T state. F6P is the other substrate for PFK-1 and it also binds preferentially to the R state enzyme. At high concentrations of ATP, the inhibitor site becomes occupied and shifting the equilibrium of PFK-1 comformation to that of the T state decreasing PFK-1's ability to bind F6P. The inhibition of PFK-1 by ATP is overcome by AMP which binds to the R state of the enzyme and, therefore, stabilizes the conformation of the enzyme capable of binding F6P. The most important allosteric regulator of both glycolysis and gluconeogenesis is fructose 2,6-bisphosphate, F2,6BP, which is not an intermediate in glycolysis or in gluconeogenesis.

Regulation of glycolysis and gluconeogenesis by fructose 2,6-bisphosphate (F2,6BP). The major sites for regulation of glycolysis and gluconeogenesis are the phosphofructokinase-1 (PFK-1) and fructose-1,6-bisphosphatase (F-1,6-BPase) catalyzed reactions. PFK-2 is the kinase activity and F-2,6-BPase is the phosphatase activity of the bi-functional regulatory enzyme, phosphofructokinase-2/fructose-2,6-

bisphosphatase. PKA is cAMP-dependent protein kinase which phosphorylates PFK-2/F-2,6-BPase turning on the phosphatase activity. (+ve) and (-ve) refer to positive and negative activities, respectively.

The synthesis of F2,6BP is catalyzed by the bifunctional enzyme phosphofructokinase-2/fructose-2,6-bisphosphatase (PFK-2/F-2,6-BPase). In the nonphosphorylated form the enzyme is known as PFK-2 and serves to catalyze the synthesis of F2,6BP by phosphorylating fructose 6-phosphate. The result is that the activity of PFK-1 is greatly stimulated and the activity of F-1,6-BPase is greatly inhibited. Under conditions where PFK-2 is active, fructose flow through the PFK-1/F-1,6-BPase reactions takes place in the glycolytic direction, with a net production of F1,6BP. When the bifunctional enzyme is phosphorylated it no longer exhibits kinase activity, but a new active site hydrolyzes F2,6BP to F6P and inorganic phosphate. The metabolic result of the phosphorylation of the bifunctional enzyme is that allosteric stimulation of PFK-1 ceases, allosteric inhibition of F-1,6-BPase is eliminated, and net flow of fructose through these two enzymes is gluconeogenic, producing F6P and eventually glucose. The interconversion of the bifunctional enzyme is catalyzed by cAMP-dependent protein kinase (PKA), which in turn is regulated by circulating peptide hormones. When blood glucose levels drop, pancreatic insulin production falls, glucagon secretion is stimulated, and circulating glucagon is highly increased. Hormones such as glucagon bind to plasma membrane receptors on liver cells, activating membrane-localized adenylate cyclase leading to an increase in the conversion of ATP to cAMP. cAMP binds to the regulatory subunits of PKA, leading to release and activation of the catalytic subunits. PKA phosphorylates numerous enzymes, including the bifunctional PFK-2/F-2,6-BPase. Under these conditions the liver stops consuming glucose and becomes metabolically gluconeogenic, producing glucose to reestablish normoglycemia. Regulation of glycolysis also occurs at the step catalyzed by pyruvate kinase, (PK). The liver enzyme has been most studied in vitro. This enzyme is inhibited by ATP and acetyl-CoA and is activated by F1,6BP. The inhibition of PK by ATP is similar to the effect of ATP on PFK-1. The binding of ATP to the inhibitor site reduces its affinity for PEP. The liver enzyme is also controlled at the level of synthesis. Increased carbohydrate ingestion induces the synthesis of PK resulting in elevated cellular levels of the enzyme. A number of PK isozymes have been described. The liver isozyme (L-type), characteristic of a gluconeogenic tissue, is regulated via phosphorylation by PKA, whereas the M-type isozyme found in brain, muscle, and other glucose requiring tissue is unaffected by PKA. As a consequence of these differences, blood glucose levels and associated hormones can regulate the balance of liver gluconeogenesis and glycolysis while muscle metabolism remains unaffected. In erythrocytes, the fetal PK isozyme has much greater activity than the adult isozyme; as a result, fetal erythrocytes have comparatively low concentrations of glycolytic intermediates. Because of the low steady-state concentration of fetal 1,3BPG, the 2,3BPG shunt is greatly reduced in fetal cells and little 2,3BPG is

formed. Since 2,3BPG is a negative effector of hemoglobin affinity for oxygen, fetal erythrocytes have a higher oxygen affinity than maternal erythrocytes. Therefore, transfer of oxygen from maternal hemoglobin to fetal hemoglobin is favored, assuring the fetal oxygen supply. In the newborn, an erythrocyte isozyme of the M-type with comparatively low PK activity displaces the fetal type, resulting in an accumulation of glycolytic intermediates. The increased 1,3BPG levels activate the 2,3BPG shunt, producing 2,3BPG needed to regulate oxygen binding to hemoglobin. Genetic diseases of adult erythrocyte PK are known in which the kinase is virtually inactive. The erythrocytes of affected individuals have a greatly reduced capacity to make ATP and thus do not have sufficient ATP to perform activities such as ion pumping and maintaining osmotic balance. These erythrocytes have a short half-life, lyse readily, and are responsible for some cases of hereditary hemolytic anemia. The liver PK isozyme is regulated by phosphorylation, allosteric effectors, and modulation of gene expression. The major allosteric effectors are F1,6BP, which stimulates PK activity by decreasing its Km(app) for PEP, and for the negative effector, ATP. Expression of the liver PK gene is strongly influenced by the quantity of carbohydrate in the diet, with high-carbohydrate diets inducing up to 10-fold increases in PK concentration as compared to low carbohydrate diets. Liver PK is phosphorylated and inhibited by PKA, and thus it is under hormonal control similar to that described earlier for PFK-2. Muscle PK (M-type) is not regulated by the same mechanisms as the liver enzyme. Extracellular conditions that lead to the phosphorylation and inhibition of liver PK, such as low blood glucose and high levels of circulating glucagon, do not inhibit the muscle enzyme. The result of this differential regulation is that hormones such as glucagon and epinephrine favor liver gluconeogenesis by inhibiting liver glycolysis, while at the same time, muscle glycolysis can proceed in accord with needs directed by intracellular conditions. back to the top

Metabolic Fates of Pyruvate

Pyruvate is the branch point molecule of glycolysis. The ultimate fate of pyruvate depends on the oxidation state of the cell. In the reaction catalyzed by G3PDH a molecule of NAD+ is reduced to NADH. In order to maintain the re-dox state of the cell, this NADH must be re-oxidized to NAD+. During aerobic glycolysis this occurs in the mitochondrial electron transport chain generating ATP. Thus, during aerobic glycolysis ATP is generated from oxidation of glucose directly at the PGK and PK reactions as well as indirectly by re-oxidation of NADH in the oxidative phosphorylation pathway. Additional NADH molecules are generated during the complete aerobic oxidation of pyruvate in the TCA cycle. Pyruvate enters the TCA cycle in the form of acetyl-CoA which is the product of the pyruvate dehydrogenase reaction. The fate of pyruvate during anaerobic glycolysis is reduction to lactate. back to the top

Lactate Metabolism During anaerobic glycolysis, that period of time when glycolysis is proceeding at a high rate (or in anaerobic organisms), the oxidation of NADH occurs through the reduction of an organic substrate. Erythrocytes and skeletal muscle (under conditions of exertion) derive all of their ATP needs through anaerobic glycolysis. The large quantity of NADH produced is oxidized by reducing pyruvate to lactate. This reaction is carried out by lactate dehydrogenase, (LDH). The lactate produced during anaerobic glycolysis diffuses from the tissues and is transproted to highly aerobic tissues such as cardiac muscle and liver. The lactate is then oxidized to pyruvate in these cells by LDH and the pyruvate is further oxidized in the TCA cycle. If the energy level in these cells is high the carbons of pyruvate will be diverted back to glucose via the gluconeogenesis pathway. Mammalian cells contain two distinct types of LDH subunits, termed M and H. Combinations of these different subunits generates LDH isozymes with different characteristics. The H type subunit predominates in aerobic tissues such as heart muscle (as the H4 tetramer) while the M subunit predominates in anaerobic tissues such as skeletal muscle as the M4 tetramer). H4 LDH has a low Km for pyruvate and also is inhibited by high levels of pyruvate. The M4 LDH enzyme has a high Km for pyruvate and is not inhibited by pyruvate. This suggsts that the H-type LDH is utilized for oxidizing lactate to pyruvate and the M-type the reverse. back to the top

Ethanol Metabolism

Animal cells (primarily hepatocytes) contain the cytosolic enzyme alcohol dehydrogenase (ADH) which oxidizes ethanol to acetaldehyde. Acetaldehyde then enters the mitochondria where it is oxidized to acetate by acetaldehyde dehydrogenase (AcDH).

Acetaldehyde forms adducts with proteins, nucleic acids and other compounds, the results of which are the toxic side effects (the hangover) that are associated with alcohol consumption. The ADH and AcDH catalyzed reactions also leads to

the reduction of NAD+ to NADH. The metabolic effects of ethanol intoxication stem from the actions of ADH and AcDH and the resultant cellular imbalance in the NADH/NAD+. The NADH produced in the cytosol by ADH must be reduced back to NAD+ via either the malate-aspartate shuttle or the glycerol-phosphate

shuttle. Thus, the ability of an individual to metabolize ethanol is dependent upon the capacity of hepatocytes to carry out eother of these 2 shuttles, which in turn

is affected by the rate of the TCA cycle in the mitochondria whose rate of function is being impacted by the NADH produced by the AcDH reaction. The reduction in

NAD+ impairs the flux of glucose through glycolysis at the glyceraldehyde-3-phosphate dehydrogenase reaction, thereby limiting energy production.

Additionally, there is an increased rate of hepatic lactate production due to the effect of increased NADH on direction of the hepatic lactate dehydrogenase

(LDH) reaction. This reverseral of the LDH reaction in hepatocytes diverts pyruvate from gluconeogenesis leading to a reduction in the capacity of the liver

to deliver glucose to the blood. In addition to the negative effects of the altered NADH/NAD+ ratio on hepatic gluconeogenesis, fatty acid oxidation is also reduced as this process requires

NAD+ as a cofactor. In fact the opposite is true, fatty acid synthesis is increased and there is an increase in triacylglyceride production by the liver. In the

mitocondria, the production of acetate from acetaldehyde leads to increased levels of acetyl-CoA. Since the increased generation of NADH also reduces the activity of the TCA cycle, the acetyl-CoA is diverted to fatty acid synthesis. The reduction in cytosolic NAD+ leads to reduced activity of glycerol-3-phosphate

dehydrogenase (in the glcerol 3-phosphate to DHAP direction) resulting in increased levels of glycerol 3-phosphate which is the backbone for the synthesis of the triacylglycerides. Both of these two events lead to fatty acid deposition in

the liver leading to fatty liver syndrome.

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Regulation of Blood Glucose Levels If for no other reason, it is because of the demands of the brain for oxidizable

glucose that the human body exquisitely regulates the level of glucose circulating in the blood. This level is maintained in the range of 5mM.

Nearly all carbohydrates ingested in the diet are converted to glucose following transport to the liver. Catabolism of dietary or cellular proteins generates carbon

atoms that can be utilized for glucose synthesis via gluconeogenesis. Additionally, other tissues besides the liver that incompletely oxidize glucose (predominantly skeletal muscle and erythrocytes) provide lactate that can be

converted to glucose via gluconeogenesis. Maintenance of blood glucose homeostasis is of paramount importance to the survival of the human organism. The predominant tissue responding to signals

that indicate reduced or elevated blood glucose levels is the liver. Indeed, one of the most important functions of the liver is to produce glucose for the circulation.

Both elevated and reduced levels of blood glucose trigger hormonal responses to initiate pathways designed to restore glucose homeostasis. Low blood glucose

triggers release of glucagon from pancreatic α-cells. High blood glucose triggers release of insulin from pancreatic β-cells. Additional signals, ACTH and growth hormone, released from the pituitary act to increase blood glucose by inhibiting

uptake by extrahepatic tissues. Glucocorticoids also act to increase blood glucose levels by inhibiting glucose uptake. Cortisol, the major glucocorticoid

released from the adrenal cortex, is secreted in response to the increase in

circulating ACTH. The adrenal medullary hormone, epinephrine, stimulates production of glucose by activating glycogenolysis in response to stressful

stimuli. Glucagon binding to its' receptors on the surface of liver cells triggers an increase in cAMP production leading to an increased rate of glycogenolysis by activating

glycogen phosphorylase via the PKA-mediated cascade. This is the same response hepatocytes have to epinephrine release. The resultant increased levels of G6P in hepatocytes is hydrolyzed to free glucose, by glucose-6-

phosphatase, which then diffuses to the blood. The glucose enters extrahepatic cells where it is re-phosphorylated by hexokinase. Since muscle and brain cells lack glucose-6-phosphatase, the glucose-6-phosphate product of hexokinase is

retained and oxidized by these tissues. In opposition to the cellular responses to glucagon (and epinephrine on

hepatocytes), insulin stimulates extrahepatic uptake of glucose from the blood and inhibits glycogenolysis in extrahepatic cells and conversely stimulates

glycogen synthesis. As the glucose enters hepatocytes it binds to and inhibits glycogen phosphorylase activity. The binding of free glucose stimulates the de-

phosphorylation of phosphorylase thereby, inactivating it. Why is it that the glucose that enters hepatocytes is not immediately phosphorylated and oxidized?

Liver cells contain an isoform of hexokinase called glucokinase. Glucokinase has a much lower affinity for glucose than does hexokinase. Therefore, it is not

fully active at the physiological ranges of blood glucose. Additionally, glucokinase is not inhibited by its product G6P, whereas, hexokinase is inhibited by G6P.

One major response of non-hepatic tissues to insulin is the recruitment, to the cell surface, of glucose transporter complexes. Glucose transporters comprise a

family of five members, GLUT-1 to GLUT-5. GLUT-1 is ubiquitously distributed in various tissues. GLUT-2 is found primarily in intestine, kidney and liver. GLUT-3 is also found in the intestine and GLUT-5 in the brain and testis. Insulin-sensitive tissues such as skeletal muscle and adipose tissue contain GLUT-4. When the concentration of blood glucose increases in response to food intake, pancreatic

GLUT-2 molecules mediate an increase in glucose uptake which leads to increased insulin secretion.

Hepatocytes, unlike most other cells, are freely permeable to glucose and are, therefore, essentially unaffected by the action of insulin at the level of increased glucose uptake. When blood glucose levels are low the liver does not compete

with other tissues for glucose since the extrahepatic uptake of glucose is stimulated in response to insulin. Conversely, when blood glucose levels are high

extrahepatic needs are satisfied and the liver takes up glucose for conversion into glycogen for future needs. Under conditions of high blood glucose, liver

glucose levels will be high and the activity of glucokinase will be elevated. The G6P produced by glucokinase is rapidly converted to G1P by

phosphoglucomutase, where it can then be incorporated into glycogen. back to the top




• Fructose Metabolism • Clinical Significances of Fructose Metabolism • Galactose Metabolism • Clinical Significances of Galactose Metabolism • Mannose Metabolism • Glycerol Metabolism • Glucuronate Metabolism • Clinical Significances of Glucuronate


Fructose Metabolism

Diets containing large amounts of sucrose (a disaccharide of glucose and fructose) can utilize the fructose as a major source of energy. The pathway to utilization of fructose differs in muscle and liver. Muscle which contains only hexokinase can phosphorylate fructose to F6P which is a direct glycolytic intermediate. In the liver which contains mostly glucokinase, which is specific for glucose as its substrate, requires the function of additional enzymes to utilize fructose in glycolysis. Hepatic fructose is phosphorylated on C-1 by fructokinase yielding fructose-1-phosphate (F1P). In liver the form of aldolase that predominates (aldolase B) can utilize both F-1,6-BP and F1P as substrates. Therefore, when presented with F1P the enzyme generates DHAP and glyceraldehyde. The DHAP is converted, by triose phosphate isomerase, to G3P and enters glycolysis. The glyceraldehyde can be phosphorylated to G3P by glyceraldehyde kinase or converted to DHAP through the concerted actions of alcohol dehydrogenase, glycerol kinase and glycerol phosphate dehydrogenase.

Entry of fructose carbon atoms into the glycolytic pathway in hepatocytes.

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Clinical Significances of Fructose Metabolism

Three inherited abnormalities in fructose metabolism have been identified. Essential fructosuria is a benign metabolic disorder caused by the lack of fructokinase which is normally present in the liver, pancreatic islets and kidney cortex. The fructosuria of this disease depends on the time and amount of fructose and sucrose intake. Since the disorder is asymptomatic and harmless it may go undiagnosed. Hereditary fructose intolerance is a potentially lethal disorder resulting from a lack of aldolase B which is normally present in the liver, small intestine and kidney cortex. The disorder is characterized by severe hypoglycemia and vomiting following fructose intake. Prolonged intake of fructose by infants with this defect leads to vomiting, poor feeding, jaundice, hepatomegaly, hemorrhage and eventually hepatic failure and death. The hypoglycemia that result following fructose uptake is caused by fructose-1-phosphate inhibition of glycogenolysis, by interfering with the phosphorylase reaction, and inhibition of gluconeogenesis at the deficient aldolase step. Patients remain symptom free on a diet devoid of fructose and sucrose. Hereditary fructose-1,6-bisphosphatase deficiency results in severely impaired hepatic gluconeogenesis and leads to episodes of hypoglycemia, apnea,

hyperventillation, ketosis and lactic acidosis. These symptoms can take on a lethal course in neonates. Later in life episodes are triggered by fasting and febrile infections. back to the top

Galactose Metabolism

Galactose, which is metabolized from the milk sugar, lactose (a disaccharide of glucose and galactose), enters glycolysis by its conversion to glucose-1-phosphate (G1P). This occurs through a series of steps. First the galactose is phosphorylated by galactokinase to yield galactose-1-phosphate. Epimerization of galactose-1-phosphate to G1P requires the transfer of UDP from uridine diphosphoglucose (UDP-glucose) catalyzed by galactose-1-phosphate uridyl transferase. This generates UDP-galactose and G1P. The UDP-galactose is epimerized to UDP-glucose by UDP-galactose-4 epimerase. The UDP portion is exchanged for phosphate generating glucose-1-phosphate which then is converted to G6P by phosphoglucose mutase.

Entry of galactose carbon atoms into the glycolytic pathway. The full name for the enzyme UDP-Glc pyrophos. is UDP-glucose pyrophosphorylase, that of UDP-Glc:Gal-1-P uridylyltransferase is UDP-glucose:αααα-D-galactose-1-phosphate uridylyltransferase.

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Clinical Significances of Galactose Metabolism

Three inherited disorders of galactose metabolism have been delineated. Classic galactosemia is a major symptom of two enzyme defects. One results from loss of the enzyme galactose-1-phosphate uridyl transferase. The second form of galactosemia results from a loss of galactokinase. These two defects are manifest by a failure of neonates to thrive. Vomiting and diarrhea occur following ingestion of milk, hence individuals are termed lactose intolerant. Clinical findings of these disorders include impaired liver function (which if left untreated leads to severe cirrhosis), elevated blood galactose, hypergalactosemia, hyperchloremic metabolic acidosis, urinary galactitol excretion and hyperaminoaciduria. Unless controlled by exclusion of galactose from the diet, these galactosemias can go on to produce blindness and fatal liver damage. Even on a galactose-restricted diet, transferase-deficient individuals exhibit urinary galacitol excretion and persistently elevated erythrocyte galactose-1-phosphate levels. Blindness is due to the conversion of circulating galactose to the sugar alcohol galacitol, by an NADPH-dependent galactose reductase that is present in neural tissue and in the lens of the eye. At normal circulating levels of galactose this enzyme activity causes no pathological effects. However, a high concentration of galacitol in the lens causes osmotic swelling, with the resultant formation of cataracts and other symptoms. The principal treatment of these disorders is to eliminate lactose from the diet. The third disorder of galactose metabolism result from a deficiency of UDP-galactose-4-epimerase. Two different forms of this deficiency have been found. One is benign affecting only red and white blood cells. The other affects multiple tissues and manifests symptoms similar to the transferase deficiency. Treatment involves restriction of dietary galactose. back to the top

Mannose Metabolism

The digestion of many polysaccharides and glycoproteins yields mannose which is phosphorylated by hexokinase to generate mannose-6-phosphate. Mannose-6-phosphate is converted to fructose-6-phosphate, by the enzyme phosphomannose isomerase, and then enters the glycolytic pathway or is converted to glucose-6-phosphate by the gluconeogenic pathway of hepatocytes. back to the top

Glycerol Metabolism

The predominant source of glycerol is adipose tissue. This molecule is the backbone for the triacylglycerols. Following release of the fatty acid portions of triacylglycerols the glycerol backbone is transported to the liver where it it phosphorylated by glycerol kinase yielding glycerol-3-phosphate. Glycerol-3-phosphate is oxidized to DHAP by glycerol-3-phosphate dehydrogenase.

DHAP then enters the glycolytic if the liver cell needs energy. However, the more likely fate of glycerol is to enter the gluconeogenesis pathway in order for the liver to produce glucose for use by the rest of the body. back to the top

Glucuronate Metabolism

Glucuronate is a highly polar molecule which is incorporated into proteoglycans as well as combining with bilirubin and steroid hormones; it can also be combined with certain drugs to increase their solubility. Glucuronate is derived from glucose in the uronic acid pathway.

The uronic acid pathway is utilized to synthesize UDP-glucuronate, glucuronate and L-ascorbate. The pathway involves the oxidation of glucosae-6-phosphate to UDP-glucuronate. The oxidation is uncoupled from energy production. UDP-glucuronate is used in the synthesis of glycosaminoglycan and proteoglycans as well as forming complexes with bilirubin, steroids and certain drugs. The glucuronate complexes form to solubilize compounds for excretion. The synthesis of ascorbate (vitamin C) does not occur in primates.

The uronic acid pathway is an alternative pathway for the oxidation of glucose that does not provide a means of producing ATP, but is utilized for the generation of the activated form of glucuronate, UDP-glucuronate. The uronic acid pathway of glucose conversion to glucuronate begins by conversion of glucose-6-phosphate is to glucose-1-phosphate by phosphoglucomutase, and then activated to UDP-glucose by UDP-glucose pyrophosphorylase. UDP-glucose

is oxidized to UDP-glucuronate by the NAD+-requiring enzyme, UDP-glucose dehydrogenase. UDP-glucuronate then serves as a precursor for the synthesis of iduronic acid and UDP-xylose and is incorporated into proteoglycans and glycoproteins or forms conjugates with bilirubin, steroids, xenobiotics, drugs and many compounds containing hydroxyl (-OH) groups. back to the top

Clinical Significance of Glucuronate

In the adult human, a significant number of erythrocytes die each day. This turnover releases significant amounts of the iron-free portion of heme, porphyrin, which is subsequently degraded. The primary sites of porphyrin degradation are found in the reticuloendothelial cells of the liver, spleen and bone marrow. The breakdown of porphyrin yields bilirubin, a product that is non-polar and therefore, insoluble. In the liver, to which is transported in the plasma bound to albumin, bilirubin is solubilized by conjugation to glucuronate. The soluble conjugated bilirubin diglucuronide is then secreted into the bile. An inability to conjugate bilirubin, for instance in hepatic disease or when the level of bilirubin production exceeds the capacity of the liver, is a contributory cause of jaundice. The conjugation of glucuronate to certain non-polar drugs is important for their solubilization in the liver. Glucuronate conjugated drugs are more easily cleared from the blood by the kidneys for excretion in the urine. The glucuronate-drug conjugation system can, however, lead to drug resistance; chronic exposure to certain drugs, such as barbiturates and AZT, leads to an increase in the synthesis of the UDP-glucuronyltransferases in the liver that are involved in glucuronate-drug conjugation. The increased levels of these hepatic enzymes result in a higher rate of drug clearance leading to a reduction in the effective dose of glucuronate cleared drugs. back to the top

return to Glycolysis Page




• Introduction

• Glycogen Breakdown • Regulation of Glycogen Catabolism • Glycogen Synthesis • Regulation of Glycogen Synthesis • Clinical Significances of Glycogen Metabolism • Table of Glycogen Storage Diseases


Introduction

Stores of readily available glucose to supply the tissues with an oxidizable energy source are found principally in the liver, as glycogen. A second major source of stored glucose is the glycogen of skeletal muscle. However, muscle glycogen is not generally available to other tissues, because muscle lacks the enzyme glucose-6-phosphatase. The major site of daily glucose consumption (75%) is the brain via aerobic pathways. Most of the remainder of is utilized by erythrocytes, skeletal muscle, and heart muscle. The body obtains glucose either directly from the diet or from amino acids and lactate via gluconeogenesis. Glucose obtained from these two primary sources either remains soluble in the body fluids or is stored in a polymeric form, glycogen. Glycogen is considered the principal storage form of glucose and is found mainly in liver and muscle, with kidney and intestines adding minor storage sites. With up to 10% of its weight as glycogen, the liver has the highest specific content of any body tissue. Muscle has a much lower amount of glycogen per unit mass of tissue, but since the total mass of muscle is so much greater than that of liver, total glycogen stored in muscle is about twice that of liver. Stores of glycogen in the liver are considered the main buffer of blood glucose levels. back to the top

Glycogenolysis

Degradation of stored glycogen (glycogenolysis) occurs through the action of glycogen phosphorylase. The action of phosphorylase is to phosphorolytically remove single glucose residues from α-(1,4)-linkages within the glycogen molecules. The product of this reaction is glucose-1-phosphate. The advantage of the reaction proceeding through a phosphorolytic step is that:

• 1. The glucose is removed from glycogen is an activated state, i.e. phosphorylated and this occurs without ATP hydrolysis.

• 2. The concentration of Pi in the cell is high enough to drive the equilibrium of the reaction the favorable direction since the free energy change of the standard state reaction is positive.

The glucose-1-phosphate produced by the action of phosphorylase is converted to glucose-6-phosphate by phosphoglucomutase: this enzyme, like phosphoglycerate mutase (of glycolysis), contains a phosphorylated amino acid in the active site (in the case of phosphoglucomutase it is a Ser residue). The enzyme phosphate is transferred to C-6 of glucose-1-phosphate generating glucose-1,6-phosphate as an intermediate. The phosphate on C-1 is then transferred to the enzyme regenerating it and glucose-6-phospahte is the released product. As mentioned above the phosphorylase mediated release of glucose from glycogen yields a charged glucose residue without the need for hydrolysis of ATP. An additional necessity of releasing phosphorylated glucose from glycogen ensures that the glucose residues do not freely diffuse from the cell. In the case of muscle cells this is acutely apparent since the purpose in glycogenolysis in muscle cells is to generate substrate for glycolysis. The conversion of glucose-6-phosphate to glucose, which occurs in the liver, kidney and intestine, by the action of glucose-6-phosphatase does not occur in skeletal muscle as these cells lack this enzyme. Therefore, any glucose released from glycogen stores of muscle will be oxidized in the glycolytic pathway. In the liver the action of glucose-6-phosphatase allows glycogenolysis to generate free glucose for maintaining blood glucose levels. Glycogen phosphorylase cannot remove glucose residues from the branch points (α-1,6 linkages) in glycogen. The activity of phosphorylase ceases 4 glucose residues from the branch point. The removal of the these branch point glucose residues requires the action of debranching enzyme (also called glucan transferase) which contains 2 activities: glucotransferase and glucosidase. The transferase activity removes the terminal 3 glucose residues of one branch and attaches them to a free C-4 end of a second branch. The glucose in α-(1,6)-linkage at the branch is then removed by the action of glucosidase. This glucose residue is uncharged since the glucosidase-catalyzed reaction is not phosphorylytic. This means that theroretically glycogenolysis occurring in skeletal muscle could generate free glucose which could enter the blood stream. However, the activity of hexokinase in muscle is so high that any free glucose is immediately phosphorylated and enters the glycolytic pathway. Indeed, the precise reason for the temporary appearance of the free glucose from glycogen is the need of the skeletal muscle cell to generate energy from glucose oxidation, thereby, precluding any chance of the glucose entering the blood. back to the top

Regulation of Glycogenolysis

Glycogen phosphorylase is a homodimeric enzyme that exist in two distinct conformational states: a T (for tense, less active) and R (for relaxed, more active) state. Phosphorylase is capable of binding to glycogen when the enzyme is in the R state. This conformation is enhanced by binding of AMP and inhibited by binding ATP or glucose-6-phosphate. The enzyme is also subject to covalent modification by phosphorylation as a means of regulating its activity. The relative activity of the un-modified phosphorylase enzyme (given the name

phosphorylase-b) is sufficient to generate enough glucose-1-phosphate for entry into glycolysis for the production of sufficient ATP to maintain the normal resting activity of the cell. This is true in both liver and muscle cells.

Pathways involved in the regulation of glycogen phosphorylase. See the text for details of the regulatory mechanisms. PKA is cAMP-dependent protein kinase. PPI-1 is phosphoprotein phosphatase-1 inhibitor. Whether a factor has positive (+ve) or negative (-ve) effects on any enzyme is indicated. Briefly, phosphorylase b is phosphorylated, and rendered highly active, by phosphorylase kinase. Phosphorylase kinase is itself phosphorylated, leading to increased activity, by PKA (itself activated through receptor mediated mechanisms). PKA also phosphorylates PPI-1 leading to an inhibition of phosphate removal allowing the activated enzymes to remain so longer. Calcium ions can activate phosphorylase kinase even in the absence of the enzyme being phosphorylated. This allows neuromuscular stimulation by acetylcholine to lead to increased glycogenolysis in the absence of receptor stimulation.

In response to lowered blood glucose the α cells of the pancreas secrete glucagon which binds to cell surface receptors on liver and several other cells. Liver cells are the primary target for the action of this peptide hormone. The response of cells to the binding of glucagon to its cell surface receptor is the activation of the enzyme adenylate cyclase which is associated with the receptor. Activation of adenylate cyclase leads to a large increase in the

formation of cAMP. cAMP binds to an enzyme called cAMP-dependent protein kinase, PKA. Binding of cAMP to the regulatory subunits of PKA leads to the release and subsequent activation of the catalytic subunits. The catalytic subunits then phosphorylate a number of proteins on serine and threonine residues. Of significance to this discussion is the PKA-mediated phosphorylation of phosphorylase kinase as shown in the diagram above. Phosphorylation of phosphorylase kinase activates the enzyme which in turn phosphorylates the b form of phosphorylase. Phosphorylation of phosphorylase-b greatly enhances its activity towards glycogen breakdown. The modified enzyme is called phosphorylase-a. The net result is an extremely large induction of glycogen breakdown in response to glucagon binding to cell surface receptors. This identical cascade of events occurs in skeletal muscle cells as well. However, in these cells the induction of the cascade is the result of epinephrine binding to receptors on the surface of muscle cells. Epinephrine is released from the adrenal glands in response to neural signals indicating an immediate need for enhanced glucose utilization in muscle, the so called fight or flight response. Muscle cells lack glucagon receptors. The presence of glucagon receptors on muscle cells would be futile anyway since the role of glucagon release is to increase blood glucose concentrations and muscle glycogen stores cannot contribute to blood glucose levels. Regulation of phosphorylase kinase activity is also affected by two distinct mechanisms involving Ca2+ ions. The ability of Ca2+ ions to regulate phosphorylase kinase is through the function of one of the subunits of this enzyme. One of the subunits of this enzyme is the ubiquitous protein, calmodulin. Calmodulin is a calcium binding protein. Binding induces a conformational change in calmodulin which in turn enhances the catalytic activity of the phosphorylase kinase towards its substrate, phosphorylase-b. This activity is crucial to the enhancement of glycogenolysis in muscle cells where muscle contraction is stimulated acetylcholine stimulation of neuromuscular junctions. The effect of acetylcholine release from nerve terminals at a neuromuscular junction is to depolarize the muscle cell leading to increased release of sarcoplasmic Ca2+, thereby activating phosphorylase kinase.Thus, not only does the increased intracellular calcium increase the rate of muscle contraction it increases glycogenolysis which provides the muscle cell with the increased ATP it also needs for contraction. The second Ca2+ ion-mediated pathway to phosphorylase kinase activation is through activation of α-adrenergic receptors by epinephrine.

Pathways involved in the regulation of glycogen phosphorylase by epinephrine activation of α-adrenergic receptors. See the text for details of the regulatory mechanisms. PLC-γ is phospholipase C-γγγγ. The substrate for PLC-γ is phosphatidylinositol-4,5-bisphosphate (PIP2) and the products are IP3, inositol trisphosphate and DAG, diacylglycerol.

Unlike β-adrenergic receptors which are coupled to activation of adenylate cyclase, α-adrenergic receptors are coupled through G-proteins that activate phospholipase-C-γγγγ (PLC-γγγγ). Activation pf PLC-γ leads to increased hydrolysis of membrane phosphatidylinositol-4,5-bisphosphate (PIP2), the products of which are inositol trisphosphate (IP3) and diacylglycerol (DAG). DAG binds to and activates protein kinase C (PKC) an enzyme that phosphorylates numerous substrate, one of which is glycogen synthase (see below). IP3 binds to receptors on the surface of the endoplasmic reticulum leading to release of Ca2+ ions. The Ca2+ ions then interact the calmodulin subunits of phosphoryase kinase resulting in its' activation. Additionally, the Ca2+ ions activate PKC in conjunction with DAG. In order to terminate the activity of the enzymes of the glycogen phosphorylase activation cascade, once the needs of the body are met, the modified enzymes need to be un-modified. In the case of Ca2+ induced activation, the level of Ca2+ ion release from muscle stores will terminate when the incoming nerve impulses cease. The removal of the phosphates on phosphorylase kinase and phosphorylase-a is carried out by phosphoprotein phosphatase-1 (PP-1). In

order that the phosphate residues placed on these enzymes by PKA and phosphorylase kinase are not immediately removed, the activity of PP-1 must also be regulated. This is accomplished by the binding of PP-1 to phosphoprotein phosphatase inhibitor (PPI-1). This protein also is phosphorylated by PKA and dephosphorylated by PP-1 (see diagram above). The phosphorylation of PPI allows it to bind to PP-1, an activity it is incapable of carrying out when not phosphorylated. When PPI binds PP-1 its phosphorylations are removed by PP-1 but at a much reduced rate than by free PP-1 thus temporarily trapping PP-1 from other substrates. The effects of the activation of this regulatory phosphorylation cascade on the rate of glycogen synthesis is described below. back to the top

Glycogen Synthesis

Synthesis of glycogen from glucose is carried out the enzyme glycogen synthase. This enzyme utilizes UDP-glucose as one substrate and the non-reducing end of glycogen as another. The activation of glucose to be used for glycogen synthesis is carried out by the enzyme UDP-glucose pyrophosphorylase. This enzyme exchanges the phosphate on C-1 of glucose-1-phosphate for UDP. The energy of the phospho-glycosyl bond of UDP-glucose is utilized by glycogen synthase to catalyze the incorporation of glucose into glycogen. UDP is subsequently released from the enzyme. The α-1,6 branches in glucose are produced by amylo-(1,4 - 1,6)-transglycosylase, also termed the branching enzyme. This enzyme transfers a terminal fragment of 6-7 glucose residues (from a polymer at least 11 glucose residues long) to an internal glucose residue at the C-6 hydroxyl position. Until recently, the source of the first glycogen molecule that might act as a primer in glycogen synthesis was unknown. Recently it has been discovered that a protein known as glycogenin is located at the core of glycogen molecules. Glycogenin has the unusual property of catalyzing its own glycosylation, attaching C-1 of a UDP-glucose to a tyrosine residue on the enzyme. The attached glucose is believed to serve as the primer required by glycogen synthase. back to the top

Regulation of Glycogen Synthesis

Glycogen synthase ia a tetrameric enzyme consisting of 4 identical subunits. The activity of glycogen synthase is regulated by phosphorylation of serine residues in the subunit proteins. Phosphorylation of glycogen synthase reduces its activity towards UDP-glucose. When in the non-phosphorylated state, glycogen synthase does not require glucose-6-phosphate as an allosteric activator---when phosphorylated it does. The two forms of glycogen synthase are identifed by the same nomenclature as used for glycogen phosphorylase. The unphosphorylated and most active form is synthase-a and the phosphorylated glucose-6-phosphate-dependent form is synthase-b shift.

Pathways involved in the regulation of glycogen synthase. See the text for details of the regulatory mechanisms. PKA is cAMP-dependent protein kinase. PPI-1 is phosphoprotein phosphatase-1 inhibitor. Whether a factor has positive (+ve) or negative (-ve) effects on any enzyme is indicated. Briefly, glycogen synthase a is phosphorylated, and rendered much less active and requires glucose-6-phosphate to have any activity at all. Phosphorylation of glycogen synthase is accomplished by several different enzymes. The most important is synthase-phosphorylase kinase the same enzyme responsible for phosphorylation (and activation) of glycogen phosphorylase. PKA (itself activated through receptor mediated mechanisms) also phosphorylates glycogen synthase directly. The effects of PKA on PPI-1 are the same as those described above for the regulation of glycogen phosphorylase. The other enzymes shown to directly phosphorylate glycogen synthase are protein kinase C (PKC), calmodulin-dependent protein kinase, glycogen synthase kinase-3 (GSK-3) and two forms of casein kinase (CK-I and CK-II). The enzyme PKC is activated by Ca2+ ions and phospholipids, primarily diacylglycerol, DAG. DAG is formed by receptor-mediated hydrolysis of membrane phosphatidylinositol bisphosphate (PIP2).

Phosphorylation of synthase occurs primarily in response to hormonal activation of PKA. One of the major kinases active on synthase is synthase-phosphorylase kinase; the same enzyme that phosphorylates glycogen phosphorylase. However, at least 5 additional enzymes have been identified

that phosphorylate glycogen synthase directly. One of of these glycogen synthase phosphorylating enzymes is PKA itself. One important glycogen synthase phosphorylating enzyme is active independently of increases in cAMP levels. This enzyme is glycogen synthase kinase 3 (GSK-3). Each phosphorylation event occurs at distinct serine residues which can result in a progressively increased state of synthase phosphorylation. Glycogen synthase activity can also be affected by epinephrine binding to α-adrenergic receptors through a pathway like that described above for regulation of glycogen phosphorylase.

Pathways involved in the regulation of glycogen synthase by epinephrine activation of α-adrenergic receptors. See the text for details of the regulatory mechanisms. PKC is protein kinase C. PLC-γ is phospholipase C-γγγγ. The substrate for PLC-γ is phosphatidylinositol-4,5-bisphosphate (PIP2) and the products are IP3, inositol trisphosphate and DAG, diacylglycerol.

When α-adrenergic receptors are stimulated there is an increase in the activity of PLC-γ with a resultant increase in PIP2 hydrolysis. The products of PIP2 hydrolysis are DAG and IP3. As described above for glycogen phoshorylase, DAG and the Ca2+ ions released by IP3 activate PKC which phosphorylates and inactivates glycogen synthase. Additional responses of calcium are the activation of calmodulin-dependent protein kinase (calmodulin is a component

of many enzymes that are responsive to Ca2+) which also phosphorytes glycogen synthase. The effects of these phosphorylations leads to:

• 1. Decreased affinity of synthase for UDP-glucose. • 2. Decreased affinity of synthase for glucose-6-phosphate. • 3. Increased affinity of synthase for ATP and Pi.

Reconversion of synthase-b to synthase-a requires dephosphorylation. This is carried out predominately by protein phosphatase-1 (PP-1) the same phosphatase involved in dephosphorylation of phosphorylase. The activity of PP-1 is also affected by insulin. The pancreatic hormone exerts an opposing effect to that of glucagon and epinephrine. This should appear obvious since the role of insulin is to increase the uptake of glucose from the blood. back to the top

Glycogen Storage Diseases

Since glycogen molecules can become enormously large, an inability to degrade glycogen can cause cells to become pathologically engorged; it can also lead to the functional loss of glycogen as a source of cell energy and as a blood glucose buffer. Although glycogen storage diseases are quite rare, their effects can be most dramatic. The debilitating effect of many glycogen storage diseases depends on the severity of the mutation causing the deficiency. In addition, although the glycogen storage diseases are attributed to specific enzyme deficiencies, other events can cause the same characteristic symptoms. For example, Type I glycogen storage disease (von Gierke's disease) is attributed to lack of glucose-6-phosphatase. However, this enzyme is localized on the cisternal surface of the endoplasmic reticulum (ER); in order to gain access to the phosphatase, glucose-6-phosphate must pass through a specific translocase in the ER membrane. Mutation of either the phosphatase or the translocase makes transfer of liver glycogen to the blood a very limited process. Thus, mutation of either gene leads to symptoms associated with von Gierke's disease, which occurs at a rate of about 1 in 200,000 people. back to the top

Table of Glycogen Storage Diseases

Type: Name

Enzyme Affected

Primary Organ

Manifestations

Type 0 glycogen synthase liver hypoglycemia, early death,

hyperketonia

Type Ia: glucose-6- liver hepatomegaly, kidney

von Gierke's phosphatase failure, thrombocyte dysfunction

Type Ib

microsomal glucose-6-phosphate translocase

liver like Ia, also neutropenia, bacterial infections

Type Ic microsomal Pi transporter liver like Ia

Type II: Pompe's

lysosomal α-1,4-glucosidase, lysosomal acid α-glucosidase acid maltase

skeletal and cardiac muscle

infantile form = death by 2; juvenile form = myopathy; adult form = muscular dystrophy-like

Type IIIa: Cori's or Forbe's

liver and muscle debranching

enzyme

liver, skeletal and cardiac muscle

infant hepatomegaly, myopathy

Type IIIb

liver debranching enzyme

normal muscle enzyme

liver, skeletal and cardiac muscle

liver symptoms same as type IIIa

Type IV: Anderson's

branching enzyme liver, muscle hepatosplenomegaly,

cirrhosis

Type V: McArdle's

muscle phosphorylase

skeletal muscle

excercise-induced cramps and pain, myoglobinuria

Type VI: Her's

liver phosphorylase liver

hepatomegaly, mild hypoglycemia,

hyperlipidemia and ketosis, improvement with age

Type VII: Tarui's muscle PFK-1 muscle,

RBC's like V, also hemolytic anemia

Type VIb, VIII or Type

phosphorylase kinase

liver, leukocytes, like VI

IX muscle

Type XI: Fanconi-

Bickel

glucose transporter-2

(GLUT-2) liver

failure to thrive, hepatomegaly, rickets, proximal renal tubular

dysfunction

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• Introduction • Pyruvate to Phosphoenolpyruvate (PEP), Bypass 1 • Fructose-1,6-bisphosphate to Fructose-6-phosphate, Bypass 2 • Glucose-6-phosphate to Glucose (or Glycogen), Bypass 3 • Substrates for Gluconeogenesis • Regulation of Gluconeogenesis


Introduction

Gluconeogenesis is the biosynthesis of new glucose, (i.e. not glucose from glycogen). The production of glucose from other metabolites is necessary for use as a fuel source by the brain, testes, erythrocytes and kidney medulla since glucose is the sole energy source for these organs. During starvation, however, the brain can derive energy from ketone bodies which are converted to acetyl-CoA. Synthesis of glucose from three and four carbon precursors is essentially a reversal of glycolysis. The relevant features of the pathway of gluconeogenesis are diagrammed below.

The relevant reactions of gluconeogenesis are depicted. The enzymes of the 3 bypass steps are indicated in green along with phosphoglycerate kinase. This latter enzyme is included since when functioning in the gluconeogenic direction the reaction consumes energy. Gluconeogenesis from 2 moles of pyruvate to 2 moles of 1,3-bisphosphoglycerate consumes 6 moles of ATP. This makes the process of gluconeogenesis very costly from an energy standpoint considering that glycolysis to 2 moles of pyruvate only yields 2 moles of ATP. Note that several steps are required in going from 2 moles of 1,3-bisphosphoglycerate to 1 mole of fructose-1,6-bisphosphate. First there is a reversal of the glyceraldehyde-3-phosphate dehydrogenase reaction which requires a supply of NADH. When lactate is the gluconeogenic substrate the NADH is supplied by the lactate dehydrogenase reaction, and it is supplied by the malate dehydrogenase reaction when pyruvate is the substrate. Secondly, 1 mole of glyceraldehyde-3-phosphate must be isomerized to DHAP and then a mole of DHAP can be condensed to a mole of glyceraldehyde-3-phosphate to form 1

mole of fructose-1,6-bisphosphate in a reversal of the aldolase reaction. Most non-hepatic tissues lack glucose-6-phosphatase and so the glucose-6-phosphate generated in these tissues would be a substrate for glycogen synthesis. In hepatocytes the glucose-6-phosphatase reactions allows the liver to supply the blood with free glucose. Remember that due to the high Km of liver glucokinase most of the glucose will not be phosphorylated and will flow down its' concentration gradient out of hepatocytes and into the blood.

The three reactions of glycolysis that proceed with a large negative free energy change are bypassed during gluconeogenesis by using different enzymes. These three are the pyruvate kinase, phosphofructokinase-1(PFK-1) and hexokinase/glucokinase catalyzed reactions. In the liver or kidney cortex and in some cases skeletal muscle, the glucose-6-phosphate (G6P) produced by gluconeogenesis can be incorporated into glycogen. In this case the third bypass occurs at the glycogen phosphorylase catalyzed reaction. Since skeletal muscle lacks glucose-6-phosphatase it cannot deliver free glucose to the blood and undergoes gluconeogenesis exclusively as a mechanism to generate glucose for storage as glycogen. back to the top

Pyruvate to Phosphoenolpyruvate (PEP), Bypass

1 Conversion of pyruvate to PEP requires the action of two mitochondrial enzymes. The first is an ATP-requiring reaction catalyzed by pyruvate carboxylase, (PC). As the name of the enzyme implies, pyruvate is carboxylated to form oxaloacetate (OAA). The CO2 in this reaction is in the form of bicarbonate (HCO3

-) . This reaction is an anaplerotic reaction since it can be used to fill-up the TCA cycle. The second enzyme in the conversion of pyruvate to PEP is PEP carboxykinase (PEPCK). PEPCK requires GTP in the decarboxylation of OAA to yield PEP. Since PC incorporated CO2 into pyruvate and it is subsequently released in the PEPCK reaction, no net fixation of carbon occurs. Human cells contain almost equal amounts of mitochondrial and cytosolic PEPCK so this second reaction can occur in either cellular compartment. For gluconeogenesis to proceed, the OAA produced by PC needs to be transported to the cytosol. However, no transport mechanism exist for its' direct transfer and OAA will not freely diffuse. Mitochondrial OAA can become cytosolic via three pathways, conversion to PEP (as indicated above through the action of the mitochondrial PEPCK), transamination to aspartate or reduction to malate, all of which are transported to the cytosol. If OAA is converted to PEP by mitochondrial PEPCK, it is transported to the cytosol where it is a direct substrate for gluconeogenesis and nothing further is required. Transamination of OAA to aspartate allows the aspartate to be transported to the cytosol where the reverse transamination occurs yielding cytosolic OAA. This transamination reaction requires continuous transport of glutamate into, and α-ketoglutarate out of, the mitochondrion. Therefore, this

process is limited by the availability of these other substrates. Either of these latter two reactions will predominate when the substrate for gluconeogenesis is lactate. Whether mitochondrial decarboxylation or transamination occurs is a function of the availability of PEPCK or transamination intermediates. Mitochondrial OAA can also be reduced to malate in a reversal of the TCA cycle reaction catalyzed by malate dehydrogenase (MDH). The reduction of OAA to malate requires NADH, which will be accumulating in the mitochondrion as the energy charge increases. The increased energy charge will allow cells to carry out the ATP costly process of gluconeogenesis. The resultant malate is transported to the cytosol where it is oxidized to OAA by cytosolic MDH which requires NAD+ and yields NADH. The NADH produced during the cytosolic oxidation of malate to OAA is utilized during the glyceraldehyde-3-phosphate dehydrogenase reaction of glycolysis. The coupling of these two oxidation-reduction reactions is required to keep gluconeogenesis functional when pyruvate is the principal source of carbon atoms. The conversion of OAA to malate predominates when pyruvate (derived from glycolysis or amino acid catabolism) is the source of carbon atoms for gluconeogenesis. When in the cytoplasm, OAA is converted to PEP by the cytosolic version of PEPCK. Hormonal signals control the level of PEPCK protein as a means to regulate the flux through gluconeogenesis (see below). The net result of the PC and PEPCK reactions is:

Pyruvate + ATP + GTP + H2O ---> PEP + ADP + GDP + Pi + 2H+ back to the top

Fructose-1,6-bisphosphate to Fructose-6-

phosphate, Bypass 2 Fructose-1,6-bisphosphate (F1,6BP) conversion to fructose-6-phosphate (F6P) is the reverse of the rate limiting step of glycolysis. The reaction, a simple hydrolysis, is catalyzed by fructose-1,6-bisphosphatase (F1,6BPase). Like the regulation of glycolysis occurring at the PFK-1 reaction, the F1,6BPase reaction is a major point of control of gluconeogenesis (see below). back to the top

Glucose-6-phosphate (G6P) to Glucose (or

Glycogen), Bypass 3 G6P is converted to glucose through the action of glucose-6-phosphatase G6Pase). This reaction is also a simple hydrolysis reaction like that of F1,6BPase. Since the brain and skeletal muscle, as well as most non-hepatic tissues, lack G6Pase activity, any gluconeogenesis that occurs in these tissues is not utilized for blood glucose supply. In the kidney, muscle and especially the liver, G6P can be shunted toward glycogen if blood glucose levels are adequate. The reactions necessary for glycogen synthesis are an alternate bypass 3 series of reactions.

Phosphorolysis of glycogen is carried out by glycogen phosphorylase, whereas, glycogen synthesis is catalyzed by glycogen synthase. The G6P produced from gluconeogenesis can be converted to glucose-1-phosphate (G1P) by phosphoglucose mutase (PGM). G1P is then converted to UDP-glucose (the substrate for glycogen synthase) by UDP-glucose pyrophosphorylase, a reaction requiring hydrolysis of UTP. <>back to the top

Substrates for Gluconeogenesis

Lactate:

Lactate is a predominate source of carbon atoms for glucose synthesis by gluconeogenesis. During anaerobic glycolysis in skeletal muscle, pyruvate is reduced to lactate by lactate dehydrogenase (LDH). This reaction serves two critical functions during anaerobic glycolysis. First, in the direction of lactate formation the LDH reaction requires NADH and yields NAD+ which is then available for use by the glyceraldehyde-3-phosphate dehydrogenase reaction of glycolysis. These two reaction are, therefore, intimately coupled during anaerobic glycolysis. Secondly, the lactate produced by the LDH reaction is released to the blood stream and transported to the liver where it is converted to glucose. The glucose is then returned to the blood for use by muscle as an energy source and to replenish glycogen stores. This cycle is termed the Cori cycle.

The Cori cycle invloves the utilization of lactate, produced by glycolysis in non-hepatic tissues, (such as muscle and erythrocytes) as a carbon source for hepatic gluconeogenesis. In this way the liver can convert the anaerobic byproduct of glycolysis, lactate, back into more glucose for reuse by non-hepatic tissues. Note that the gluconeogenic leg of the cycle (on its own) is a net consumer of energy, costing the body 4 moles of ATP more than are produced during glycolysis. Therefore, the cycle cannot be sustained indefinitely.

Pyruvate:

Pyruvate, generated in muscle and other peripheral tissues, can be transaminated to alanine which is returned to the liver for gluconeogenesis. The transamination reaction requires an α-amino acid as donor of the amino group, generating an α-keto acid in the process. This pathway is termed the glucose-alanine cycle. Although the majority of amino acids are degraded in the liver some are deaminated in muscle. The glucose-alanine cycle is, therefore, an indirect mechanism for muscle to eliminate nitrogen while replenishing its energy supply. However, the major function of the glucose-alanine cycle is to allow non-hepatic tissues to deliver the amino portion of catabolized amino acids to the liver

for excretion as urea. Within the liver the alanine is converted back to pyruvate and used as a gluconeogenic substrate (if that is the hepatic requirement) or oxidized in the TCA cycle. The amino nitrogen is converted to urea in the urea cycle and excreted by the kidneys.

Amino Acids:

All 20 of the amino acids, excepting leucine and lysine, can be degraded to TCA cycle intermediates as discussed in the metabolism of amino acids. This allows the carbon skeletons of the amino acids to be converted to those in oxaloacetate and subsequently into pyruvate. The pyruvate thus formed can be utilized by the gluconeogenic pathway. When glycogen stores are depleted, in muscle during exertion and liver during fasting, catabolism of muscle proteins to amino acids contributes the major source of carbon for maintenance of blood glucose levels.

Glycerol:

Oxidation of fatty acids yields enormous amounts of energy on a molar basis, however, the carbons of the fatty acids cannot be utilized for net synthesis of glucose. The two carbon unit of acetyl-CoA derived from β-oxidation of fatty acids can be incorporated into the TCA cycle, however, during the TCA cycle two carbons are lost as CO2. Thus, explaining why fatty acids do not undergo net conversion to carbohydrate. The glycerol backbone of lipids can be used for gluconeogenesis. This requires phosphorylation to glycerol-3-phosphate by glycerol kinase and dehydrogenation to dihydroxyacetone phosphate (DHAP) by glyceraldehyde-3-phosphate dehydrogenase(G3PDH). The G3PDH reaction is the same as that used in the transport of cytosolic reducing equivalents into the mitochondrion for use in oxidative phosphorylation. This transport pathway is called the glycerol-phosphate shuttle. The glycerol backbone of adipose tissue stored triacylgycerols is ensured of being used as a gluconeogenic substrate since adipose cells lack glycerol kinase. In fact adipocytes require a basal level of glycolysis in order to provide them with DHAP as an intermediate in the synthesis of triacyglycerols.

Propionate:

Oxidation of fatty acids with an odd number of carbon atoms and the oxidation of some amino acids generates as the terminal oxidation product, propionyl-CoA. Propionyl-CoA is converted to the TCA intermediate, succinyl-CoA. This conversion is carried out by the ATP-requiring enzyme, propionyl-CoA carboxylase then methylmalonyl-CoA epimerase and finally the vitamin B12 requiring enzyme, methylmalonyl-CoA mutase. The utilization of propionate in gluconeogenesis only has quantitative significance in ruminants. back to the top

Regulation of Gluconeogenesis Obviously the regulation of gluconeogenesis will be in direct contrast to the regulation of glycolysis. In general, negative effectors of glycolysis are positive effectors of gluconeogenesis. Regulation of the activity of PFK-1 and F1,6BPase is the most significant site for controlling the flux toward glucose oxidation or glucose synthesis. As described in control of glycolysis, this is predominantly controlled by fructose-2,6-bisphosphate, F2,6BP which is a powerful negative allosteric effector of F1,6Bpase activity.

Regulation of glycolysis and gluconeogenesis by fructose 2,6-bisphosphate (F2,6BP). The major sites for regulation of glycolysis and gluconeogenesis are the phosphofructokinase-1 (PFK-1) and fructose-1,6-bisphosphatase (F-1,6-BPase) catalyzed reactions. PFK-2 is the kinase activity and F-2,6-BPase is the phosphatase activity of the bi-functional regulatory enzyme, phosphofructokinase-2/fructose-2,6-bisphosphatase. PKA is cAMP-dependent protein kinase which phosphorylates PFK-2/F-2,6-BPase turning on the phosphatase activity. (+ve) and (-ve) refer to positive and negative activities, respectively.

The level of F2,6BP will decline in hepatocytes in response to glucagon stimulation as well as stimulation by catecholamines. Each of these signals is elicited through activation of cAMP-dependent protein kinase (PKA). One

substrate for PKA is PFK-2, the bifunctional enzyme responsible for the synthesis and hydrolysis of F2,6BP. When PFK-2 is phosphorylated by PKA it acts as a phosphatase leading to the dephosphorylation of F2,6BP with a concomitant increase in F1,6Bpase activity and a decrease in PFK-1 activity. Secondarily, F1,6Bpase activity is regulated by the ATP/ADP ratio. When this is high, gluconeogenesis can proceed maximally. Gluconeogenesis is also controlled at the level of the pyruvate to PEP bypass. The hepatic signals elicited by glucagon or epinephrine lead to phosphorylation and inactivation of pyruvate kinase (PK) which will allow for an increase in the flux through gluconeogenesis. PK is also allosterically inhibited by ATP and alanine. The former signals adequate energy and the latter that sufficient substrates for gluconeogenesis are available. Conversely, a reduction in energy levels as evidenced by increasing concentrations of ADP lead to inhibition of both PC and PEPCK. Allosteric activation of PC occurs through acetyl-CoA. Each of these regulations occurs on a short time scale, whereas long-term regulation can be effected at the level of PEPCK. The amount of this enzyme increases in response to prolonged glucagon stimulation. This situation would occur in a starving individual or someone with an inadequate diet. back to the top




• The Pyruvate Dehydrogenase (PDH) Complex • Regulation of the PDH Complex • Reactions of the TCA Cycle • Regulation of the TCA Cycle

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The Pyruvate Dehydrogenase (PDH) Complex

The bulk of ATP used by many cells to maintain homeostasis is produced by the oxidation of pyruvate in the TCA cycle. During this oxidation process, reduced nicotinamide adenine dinucleotide (NADH) and reduced flavin adenine dinucleotide (FADH2) are generated. The NADH and FADH2 are principally

used to drive the processes of oxidative phosphorylation, which are responsible for converting the reducing potential of NADH and FADH2 to the high energy phosphate in ATP. The fate of pyruvate depends on the cell energy charge. In cells or tissues with a high energy charge pyruvate is directed toward gluconeogenesis, but when the energy charge is low pyruvate is preferentially oxidized to CO2 and H2O in the TCA cycle, with generation of 15 equivalents of ATP per pyruvate. The enzymatic activities of the TCA cycle (and of oxidative phosphorylation) are located in the mitochondrion. When transported into the mitochondrion, pyruvate encounters two principal metabolizing enzymes: pyruvate carboxylase (a gluconeogenic enzyme) and pyruvate dehydrogenase (PDH), the first enzyme of the PDH complex. With a high cell-energy charge coenzyme A (CoA) is highly acylated, principally as acetyl-CoA, and able allosterically to activate pyruvate carboxylase, directing pyruvate toward gluconeogenesis. When the energy charge is low CoA is not acylated, pyruvate carboxylase is inactive, and pyruvate is preferentially metabolized via the PDH complex and the enzymes of the TCA cycle to CO2 and H2O. Reduced NADH and FADH2 generated during the oxidative reactions can then be used to drive ATP synthesis via oxidative phosphorylation. The PDH complex is comprised of multiple copies of 3 separate enzymes: pyruvate dehydrogenase (20-30 copies), dihydrolipoyl transacetylase (60 copies) and dihydrolipoyl dehydrogenase (6 copies). The complex also requires 5 different coenzymes: CoA, NAD+, FAD+, lipoic acid and thiamine pyrophosphate (TPP) . Three of the coenzymes of the complex are tightly bound to enzymes of the complex (TPP, lipoic acid and FAD+) and two are employed as carriers of the products of PDH complex activity (CoA and NAD+). The pathway of PDH oxidation of pyruvate to acetyl-CoA is diagrammed below.

Flow diagram depicting the overall activity of the pyruvate dehydrogenase complex. During the oxidation of pyruvate to CO2 by pyruvate dehydrogenase the electrons flow from pyruvate to the lipoamide moiety of dihydrolipoyl transacetylase then to the FAD cofactor of dihydrolipoyl dehydrogenase and finally to reduction of NAD+ to NADH. The acetyl group is linked to coenzyme A (CoASH) in a high energy thioester bond. The acetyl-CoA then enters the TCA cycle for complete oxidation to CO2 and H2O.

The first enzyme of the complex is PDH itself which oxidatively decarboxylates pyruvate. During the course of the reaction the acetyl group derived from decarboxylation of pyruvate is bound to TPP. The next reaction of the complex is the transfer of the 2--carbon acetyl group from acetyl-TPP to lipoic acid, the covalently bound coenzyme of lipoyl transacetylase. The transfer of the acetyl group from acyl-lipoamide to CoA results in the formation of 2 sulfhydryl (SH) groups in lipoate requiring reoxidation to the disulfide (S-S) form to regenerate lipoate as a competent acyl acceptor. The enzyme dihydrolipoyl dehydrogenase, with FAD+ as a cofactor, catalyzes that oxidation reaction. The final activity of the PDH complex is the transfer of reducing equivalents from the

FADH2 of dihydrolipoyl dehydrogenase to NAD+. The fate of the NADH is oxidation via mitochondrial electron transport, to produce 3 equivalents of ATP: The net result of the reactions of the PDH complex are:

Pyruvate + CoA + NAD+ ------> CO2 + acetyl-CoA + NADH + H+

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Regulation of the PDH Complex

The reactions of the PDH complex serves to interconnect the metabolic pathways of glycolysis, gluconeogenesis and fatty acid synthesis to the TCA cycle. As a consequence, the activity of the PDH complex is highly regulated by a variety of allosteric effectors and by covalent modification. The importance of the PDH complex to the maintenance of homeostasis is evident from the fact that although diseases associated with deficiencies of the PDH complex have been observed, affected individuals often do not survive to maturity. Since the energy metabolism of highly aerobic tissues such as the brain is dependent on normal conversion of pyruvate to acetyl-CoA, aerobic tissues are most sensitive to deficiencies in components of the PDH complex. Most genetic diseases associated with PDH complex deficiency are due to mutations in PDH. The main pathologic result of such mutations is moderate to severe cerebral lactic acidosis and encephalopathies. The main regulatory features of the PDH complex are diagrammed below.

Factors regulating the activity of pyruvate dehydrogenase, (PDH). PDH activity is regulated by its' state of phosphorylation, being most active in the dephosphorylated state. Phosphorylation of PDH is catalyzed by a specific PDH kinase. The activity of the kinase is enhanced when cellular energy charge is high which is reflected by an increase in the level of ATP, NADH and acetyl-CoA. Conversely, an increase in pyruvate strongly inhibits PDH kinase. Additional negative effectors of PDH kinase are ADP, NAD+ and CoASH, the levels of which increase when energy levels fall. The regulation of PDH phosphatase is not completely understood but it is known that Mg2+ and Ca2+ activate the enzyme. In adipose tissue insulin increases PDH activity and in cardiac muscle PDH activity is increased by catecholamines.

Two products of the complex, NADH and acetyl-CoA, are negative allosteric effectors on PDH-a, the non-phosphorylated, active form of PDH. These effectors reduce the affinity of the enzyme for pyruvate, thus limiting the flow of carbon through the PDH complex. In addition, NADH and acetyl-CoA are powerful positive effectors on PDH kinase, the enzyme that inactivates PDH by converting it to the phosphorylated PDH-b form. Since NADH and acetyl-CoA accumulate

when the cell energy charge is high, it is not surprising that high ATP levels also up-regulate PDH kinase activity, reinforcing down-regulation of PDH activity in energy-rich cells. Note, however, that pyruvate is a potent negative effector on PDH kinase, with the result that when pyruvate levels rise, PDH-a will be favored even with high levels of NADH and acetyl-CoA. Concentrations of pyruvate which maintain PDH in the active form (PDH-a) are sufficiently high so that, in energy-rich cells, the allosterically down-regulated, high Km form of PDH is nonetheless capable of converting pyruvate to acetyl-CoA. With large amounts of pyruvate in cells having high energy charge and high NADH, pyruvate carbon will be directed to the 2 main storage forms of carbon---glycogen via gluconeogenesis and fat production via fatty acid synthesis---where acetyl-CoA is the principal carbon donor. Although the regulation of PDH-b phosphatase is not well understood, it is quite likely regulated to maximize pyruvate oxidation under energy-poor conditions and to minimize PDH activity under energy-rich conditions. back to the top

Reactions of the TCA Cycle

The TCA cycle showing enzymes, substrates and products. The abbreviated enzymes are: IDH = isocitrate dehydrogenase and α-KGDH = αααα-ketoglutarate dehydrogenase. The GTP generated during the succinate thiokinase (succinyl-CoA synthetase) reaction is equivalent to a mole of ATP by virtue of the presence of nucleoside diphosphokinase. The 3 moles of NADH and 1 mole of FADH2 generated during each round of the cycle feed into the oxidative phosphorylation pathway. Each mole of NADH leads to 3 moles of ATP and each mole of FADH2 leads to 2 moles of ATP. Therefore, for each mole of pyruvate which enters the TCA cycle, 12 moles of ATP can be generated.

Citrate Synthase (Condensing enzyme)

The first reaction of the cycle is condensation of the methyl carbon of acetyl-CoA with the keto carbon (C-2) of oxaloacetate (OAA). The standard free energy of the reaction, -8.0 kcal/mol, drives it strongly in the forward direction. Since the formation of OAA from its precursor is thermodynamically unfavorable, the highly

exergonic nature of the citrate synthase reaction is of central importance in keeping the entire cycle going in the forward direction, since it drives oxaloacetate formation by mass action principals. When the cellular energy charge increases the rate of flux through the TCA cycle will decline leading to a build-up of citrate. Excess citrate is used to transport acetyl-CoA carbons from the mitochondrion to the cytoplasm where they can be used for fatty acid and cholesterol biosynthesis. Additionally, the increased levels of citrate in the cytoplasm activate the key regulatory enzyme of fatty acid biosynthesis, acetyl-CoA carboxylase (ACC) and inhibit PFK-1. In non-hepatic tissues citrate is also required for ketone body synthesis.

Aconitase

The isomerization of citrate to isocitrate by aconitase is stereospecific, with the migration of the -OH from the central carbon of citrate (formerly the keto carbon of OAA) being always to the adjacent carbon which is derived from the methylene (-CH2-) of OAA. The stereospecific nature of the isomerization determines that the CO2 lost, as isocitrate is oxidized to succinyl-CoA, is derived from the oxaloacetate used in citrate synthesis. Aconitase is one of several mitochondrial enzymes known as non-heme-iron proteins. These proteins contain inorganic iron and sulfur, known as iron sulfur centers, in a coordination complex with cysteine sulfurs of the protein. There are two prominent classes of non-heme-iron complexes, those containing two equivalents each of inorganic iron and sulfur Fe2S2, and those containing 4 equivalents of each Fe4S4. Aconitase is a member of the Fe4S4 class. Its iron sulfur centers are often designated as Fe4S4Cys4, indicating that 4 cystine sulfur atoms are involved in tghe complete structure of the complex. In iron sulfur compounds the iron is generally involved in oxidation-reduction events.

Isocitrate Dehydrogenase

Isocitrate is oxidatively decarboxylated to α-ketoglutarate by isocitrate dehydrogenase, (IDH). There are two different IDH enzymes. The IDH of the TCA cycle uses NAD+ as a cofactor, whereas the other IDH uses NADP+ as a cofactor. Unlike the NAD+-requiring enzyme, which is located only in the mitochondrial matrix, the NADP+-requiring enzyme is found in both the mitochondrial matrix and the cytosol. IDH catalyzes the rate-limiting step, as well as the first NADH-yielding reaction of the TCA cycle. The CO2 produced by the IDH reaction is the original C-1 of the oxaloacetate used in the citrate synthase reaction. It is generally considered that control of carbon flow through the cycle is regulated at IDH by the powerful negative allosteric effectors NADH and ATP and by the potent positive effectors; isocitrate, ADP and AMP. From the latter it is clear that cell energy charge is a key factor in regulating carbon flow through the TCA cycle.

αααα-Ketoglutarate Dehydrogenase Complex

α-ketoglutarate is oxidatively decarboxylated to succinyl-CoA by the αααα-ketoglutarate dehydrogenase (αααα-KGDH) complex. This reaction generates the second TCA cycle equivalent of CO2 and NADH. This multienzyme complex is very similar to the PDH complex in the intricacy of its protein makeup, cofactors, and its mechanism of action. Also, as with the PDH complex, the reactions of the α-KGDH complex proceed with a large negative standard free energy change. Although the α-KGDH of the complex is not subject to covalent modification, allosteric regulation is quite complex, with activity being regulated by energy charge, the NAD+/NADH ratio, and effector activity of substrates and products. Succinyl-CoA and α-ketoglutarate are also important metabolites outside the TCA cycle. In particular, α-ketoglutarate represents a key anapleurotic metabolite linking the entry and exit of carbon atoms from the TCA cycle to pathways involved in amino acid metabolism. α-ketoglutarate is also important for driving the malate-aspartate shuttle. Succinyl-CoA, along with glycine, contributes all the carbon and nitrogen atoms required for the synthesis of protoporphyrin heme biosynthesis and for non-hepatic tissue utilization of ketone bodies.

Succinyl CoA Synthetase (Succinyl Thiokinase )

The conversion of succinyl-CoA to succinate by succinyl CoA synthetase involves use of the high-energy thioester of succinyl-CoA to drive synthesis of a high-energy nucleotide phosphate, by a process known as substrate-level phosphorylation. In this process a high energy enzyme--phosphate intermediate is formed, with the phosphate subsequently being transferred to GDP. Mitochondrial GTP is used in a trans-phosphorylation reaction catalyzed by the mitochondrial enzyme nucleoside diphospho kinase to phosphorylate ADP, producing ATP and regenerating GDP for the continued operation of succinyl CoA synthetase.

Succinate Dehydrogenase (SDH)

Succinate dehydrogenase catalyzes the oxidation of succinate to fumarate with the sequential reduction of enzyme-bound FAD and non-heme-iron. In mammalian cells the final electron acceptor is coenzyme Q10 (CoQ10), a mobile carrier of reducing equivalents that is restricted by its lipophilic nature to the lipid phase of the mitochondrial membrane.

Fumarase (fumarate hydratase)

The fumarase-catalyzed reactions specific for the trans form of fumarate. The result is that the hydration of fumarate proceeds stereospecifically with the production of L-malate.

Malate Dehydrogenase (MDH)

L-malate is the specific substrate for MDH, the final enzyme of the TCA cycle. The forward reaction of the cycle, the oxidation of malate yields oxaloacetate (OAA). In the forward direction the reaction has a standard free energy of about +7 kcal/mol, indicating the very unfavorable nature of the forward direction. As noted earlier, the citrate synthase reaction that condenses oxaloacetate with acetyl-CoA has a standard free energy of about -8 kcal/mol and is responsible for pulling the MDH reaction in the forward direction. The overall change in standard free energy change is about -1 kcal/mol for the conversion of malate to oxaloacetate and on to succinate. The overall stoichiometry of the TCA cycle is: acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2H2O ----> 2CO2 + 3NADH + FADH2

+ GTP + 2H+ + HSCoA

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Regulation of the TCA Cycle

Regulation of the TCA cycle. like that of glycolysis, occurs at both the level of entry of substrates into the cycle as well as at the key reactions of the cycle. Fuel enters the TCA cycle primarily as acetyl-CoA. The generation of acetyl-CoA from carbohydrates is, therefore, a major control point of the cycle. This is the reaction catalyzed by the PDH complex. By way of review, the PDH complex is inhibited by acetyl-CoA and NADH and activated by non-acetylated CoA (CoASH) and NAD+. The pyruvate dehydrogenase activities of the PDH complex are regulated by their state of phosphorylation. This modification is carried out by a specific kinase (PDH kinase) and the phosphates are removed by a specific phosphatase (PDH phosphatase). The phosphorylation of PDH inhibits its activity and, therefore, leads to decreased oxidation of pyruvate. PDH kinase is activated by NADH and acetyl-CoA and inhibited by pyruvate, ADP, CoASH, Ca2+ and Mg2+. The PDH phosphatase, in contrast, is activated by Mg2+ and Ca2+. Since three reactions of the TCA cycle as well as PDH utilize NAD+ as co-factor it is not difficult to understand why the cellular ratio of NAD+/NADH has a major impact on the flux of carbon through the TCA cycle. Substrate availability can also regulate TCA flux. This occurs at the citrate synthase reaction as a result of reduced availability of oxaloacetate. Product inhibition also controls the TCA flux, e.g. citrate inhibits citrate synthase, α-KGDH is inhibited by NADH and succinyl-CoA. The key enzymes of the TCA cycle are also regulated allosterically by Ca2+, ATP and ADP. back to the top




• Introduction • The Reactions of the PPP • Metabolic Disorders Associated with the PPP • Erythrocytes and the PPP


Introduction

The pentose phosphate pathway is primarily an anabolic pathway that utilizes the 6 carbons of glucose to generate 5 carbon sugars and reducing equivalents. However, this pathway does oxidize glucose and under certain conditions can completely oxidize glucose to CO2 and water. The primary functions of this pathway are:

• 1. To generate reducing equivalents, in the form of NADPH, for reductive biosynthesis reactions within cells.

• 2. To provide the cell with ribose-5-phosphate (R5P) for the synthesis of the nucleotides and nucleic acids.

• 3. Although not a significant function of the PPP, it can operate to metabolize dietary pentose sugars derived from the digestion of nucleic acids as well as to rearrange the carbon skeletons of dietary carbohydrates into glycolytic/gluconeogenic intermediates

Enzymes that function primarily in the reductive direction utilize the NADP+/NADPH cofactor pair as co-factors as opposed to oxidative enzymes that utilize the NAD+/NADH cofactor pair. The reactions of fatty acid biosynthesis and steroid biosynthesis utilize large amounts of NADPH. As a consequence, cells of the liver, adipose tissue, adrenal cortex, testis and lactating mammary gland have high levels of the PPP enzymes. In fact 30% of the oxidation of glucose in the liver occurs via the PPP. Additionally, erythrocytes utilize the reactions of the PPP to generate large amounts of NADPH used in the reduction of glutathione (see below). The conversion of ribonucleotides to deoxyribonucleotides (through the action of ribonucleotide reductase) requires NADPH as the electron source, therefore, any rapidly proliferating cell needs

large quantities of NADPH. back to the top

Reactions of the Pentose Phosphate Pathway

The reactions of oxidative portion of the pentose phosphate pathway are shown. The enzymes are in green.

The reactions of the non-oxidative portion of the pentose phosphate pathway are shown. Enzymes are in green. Relevant carbohydrate intermediates of this portion of the pathway are in red.

The reactions of the PPP operate exclusively in the cytoplasm. From this perspective it is understandable that fatty acid synthesis (as opposed to oxidation) takes place in the cytoplasm. The pentose phosphate pathway has both an oxidative and a non-oxidative arm. The oxidation steps, utilizing glucose-6-phosphate (G6P) as the substrate, occur at the beginning of the pathway and are the reactions that generate NADPH. The reactions catalyzed by glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase generate one mole of NADPH each for every mole of glucose-6-phosphate (G6P) that enters the PPP. The non-oxidative reactions of the PPP are primarily designed to generate R5P. Equally important reactions of the PPP are to convert dietary 5 carbon sugars into both 6 (fructose-6-phosphate) and 3 (glyceraldehyde-3-phosphate) carbon sugars which can then be utilized by the pathways of glycolysis. The primary enzymes involved in the non-oxidative steps of the PPP are transaldolase and transketolase.

• Transketolase functions to transfer 2 carbon groups from substrates of the PPP, thus rearranging the carbon atoms that enter this pathway. Like

other enzymes that transfer 2 carbon groups, transketolase requires thiamine pyrophosphate (TPP) as a co-factor in the transfer reaction.

• Transaldolase transfers 3 carbon groups and thus is also involved in a rearrangement of the carbon skeletons of the substrates of the PPP. The transaldolase reaction involves Schiff base formation between the substrate and a lysine residue in the enzyme.

The net result of the PPP, if not used solely for R5P production, is the oxidation of G6P, a 6 carbon sugar, into a 5 carbon sugar. In turn, 3 moles of 5 carbon sugar are converted, via the enzymes of the PPP, back into two moles of 6 carbon sugars and one mole of 3 carbon sugar. The 6 carbon sugars can be recycled into the pathway in the form of G6P, generating more NADPH. The 3 carbon sugar generated is glyceraldehyde-3-phsphate which can be shunted to glycolysis and oxidized to pyruvate. Alternatively, it can be utilized by the gluconeogenic enzymes to generate more 6 carbon sugars (fructose-6-phosphate or glucose-6-phosphate). back to the top

Metabolic Disorders Associated with the

Pentose Phosphate Pathway Oxidative stress within cells is controlled primarily by the action of the peptide, glutathione, GSH. See Specialized Products of Amino Acids for the synthesis of GSH.

Glutathione (GSH) is a tripeptide composed of γ-glutamate, cysteine and glycine. The sulfhydryl side chains of the cysteine residues of two glutathione molecules form a disulfide bond (GSSG) during the course of being oxidized in reactions with various oxides and peroxides in cells. Reduction of GSSG to two moles of GSH is the function of glutathione reductase, an enzyme that requires coupled oxidation of NADPH.

Glutathione is the tripeptide γγγγ-glutamylcysteinylglycine. The cysteine thiol plays the role in reducing oxidized thiols in other proteins. Oxidation of 2 cysteine thiols forms a disulfide bond. Although this bond plays a very important role in protein structure and function, inappropriately introduced disulfides can be detrimental. Glutathione can reduce disulfides nonenzymatically. Oxidative stress also generates peroxides that in turn can be reduced by glutathione to generate water and an alcohol, or 2 waters if the peroxide were hydrogen peroxide. Regeneration of reduced glutathione is carried out by the enzyme, glutathione reductase. This enzyme requires the co-factor NADPH when operating in the direction of glutathione reduction which is the thermodynamically favored direction of the reaction. It should be clear that any disruption in the level of NADPH may have a profound effect upon a cells ability to deal with oxidative stress. No other cell than the erythrocyte is exposed to greater oxidizing conditions. After all it is the oxygen carrier of the body. back to the top

Erythrocytes and the Pentose Phosphate Pathway The predominant pathways of carbohydrate metabolism in the red blood cell (RBC) are glycolysis, the PPP and 2,3-bisphosphoglycerate (2,3-BPG) metabolism (refer to discussion of hemoglobin for review of role of 2,3-BPG). Glycolysis provides ATP for membrane ion pumps and NADH for re-oxidation of methemoglobin. The PPP supplies the RBC with NADPH to maintain the reduced state of glutathione. The inability to maintain reduced glutathione in RBCs leads to increased accumulation of peroxides, predominantly H2O2, that in turn results in a weakening of the cell wall and concomitant hemolysis. Accumulation of H2O2 also leads to increased rates of oxidation of hemoglobin to methemoglobin that also weakens the cell wall. Glutathione removes peroxides via the action of glutathione peroxidase. The PPP in erythrocytes is essentially the only pathway for these cells to produce NADPH. Any defect in the production of NADPH could, therefore, have profound effects on erythrocyte survival. Several deficiencies in the level of activity (not function) of glucose-6-phosphate dehydrogenase have been observed to be associated with resistance to the malarial parasite, Plasmodium falciparum, among individuals of Mediterranean and African descent. The basis for this resistance is the weakening of the red cell

membrane (the erythrocyte is the host cell for the parasite) such that it cannot sustain the parasitic life cycle long enough for productive growth. back to the top




• Introduction • Principals of Reduction/Oxidation Reactions • Complexes of the Electron Transport Chain • Oxidative Phosphorylation • Stoichiometry of Oxidative Phosphorylation • Regulation of Oxidative Phosphorylation • Inhibitors of Oxidative Phosphaorylation • Energy from Cytosolic NADH • Other Biological Oxidations


Introduction

While the large quantity of NADH resulting from TCA cycle activity can be used for reductive biosynthesis, the reducing potential of mitochondrial NADH is most often used to supply the energy for ATP synthesis via oxidative phosphorylation. Oxidation of NADH with phosphorylation of ADP to form ATP are processes supported by the mitochondrial electron transport assembly and ATP synthase, which are integral protein complexes of the inner mitochondrial membrane. The electron transport assembly is comprised of a series of protein complexes that catalyze sequential oxidation reduction reactions; some of these reactions are thermodynamically competent to support ATP production via ATP synthase provided a coupling mechanism, such as a common intermediate, is available. Proton translocation and the development of a transmembrane proton gradient provides the required coupling mechanism. back to the top

Principals of Reduction/Oxidation (Redox) Reactions

Redox reactions involve the transfer of electrons from one chemical species to another. The oxidized plus the reduced form of each chemical species is referred to as an electrochemical half cell. Two half cells having at least one common intermediate comprise a complete, coupled, redox reaction. Coupled electrochemical half cells have the thermodynamic properties of other coupled chemical reactions. If one half cell is far from electrochemical equilibrium, its tendency to achieve equilibrium (i.e., to gain or lose electrons) can be used to alter the equilibrium position of a coupled half cell. An example of a coupled redox reaction is the oxidation of NADH by the electron transport chain:

NADH + (1/2)O2 + H+ -----> NAD+ + H2O The thermodynamic potential of a chemical reaction is calculated from equilibrium constants and concentrations of reactants and products. Because it is not practical to measure electron concentrations directly, the electron energy potential of a redox system is determined from the electrical potential or voltage of the individual half cells, relative to a standard half cell. When the reactants and products of a half cell are in their standard state and the voltage is determined relative to a standard hydrogen half cell (whose voltage, by convention, is zero), the potential observed is defined as the standard electrode potential, E0. If the pH of a standard cell is in the biological range, pH 7, its potential is defined as the standard biological electrode potential and designated E0

'. By convention, standard electrode potentials are written as potentials for reduction reactions of half cells. The free energy of a typical reaction is calculated directly from its E0

' by the Nernst equation as shown below, where n is the number of electrons involved in the reaction and F is the Faraday constant (23.06 kcal/volt/mol or 94.4 kJ/volt/mol):

∆∆∆∆G0' = -nF∆∆∆∆E0'

For the oxidation of NADH, the standard biological reduction potential is -52.6 kcal/mol. With a free energy change of -52.6 kcal/mol, it is clear that NADH oxidation has the potential for driving the synthesis of a number of ATPs since the standard free energy for the reaction:

ADP + Pi ------> ATP

is +7.3 kcal/mole. Classically, the description of ATP synthesis through oxidation of reduced electron carriers indicated 3 moles of ATP could be generated for every mole of NADH and 2 moles for every mole of FADH2. However, direct chemical analysis has shown that for every 2 electrons transferred from NADH to oxygen, 2.5 equivalents of ATP are synthesized and 1.5 for FADH2. back to the top

Complexes of the Electron Transport Chain

NADH is oxidized by a series of catalytic redox carriers that are integral proteins of the inner mitochondrial membrane. The free energy change in several of these

steps is very exergonic. Coupled to these oxidation reduction steps is a transport process in which protons (H+) from the mitochondrial matrix are translocated to the space between the inner and outer mitochondrial membranes. The redistribution of protons leads to formation of a proton gradient across the mitochondrial membrane. The size of the gradient is proportional to the free energy change of the electron transfer reactions. The result of these reactions is that the redox energy of NADH is converted to the energy of the proton gradient. In the presence of ADP, protons flow down their thermodynamic gradient from outside the mitochondrion back into the mitochondrial matrix. This process is facilitated by a proton carrier in the inner mitochondrial membrane known as ATP synthase. As its name implies, this carrier is coupled to ATP synthesis. Electron flow through the mitochondrial electron transport assembly is carried out through several enzyme complexes. Electrons enter the transport chain primarily from cytosolic NADH to mitochondrial NADH but can also be supplied by succinate (to mitochondrial FADH2) or by the glycerol phosphate shuttle via mitochondrial FADH2.

Diagrammatic representation of the flow of electrons from either NADH or succinate to oxygen (O2) in the electron transport chain of oxidative

phosphorylation. Complex I contains FMN and 22-24 iron-sulfur (Fe-S) proteins in 5-7 clusters. Complex II contains FAD and 7-8 Fe-S proteins in 3 clusters and cytochrome b560. Complex III contains cytochrome b, cytochrome c1 and one Fe-S protein. Complex IV contains cytochrome a, cytochrome a3 and 2 copper ions. As electrons pass through the proteins of complex I 4 protons (H+) are pumped into the intramembrane space of the mitochondrion. Two protons are pumped into the intramembrane space as electrons flow through complexes II, III and IV. These protons are returned to the matrix of the mitochondrion, down their concentration gradient, by passing through ATP synthase coupling electron flow and proton pumping to ATP synthesis.

With the exception of NADH, succinate, and CoQ, all of the components of the pathway are integral proteins of the inner mitochondrial membrane whose cofactors undergo redox reactions. NADH and succinate are soluble in the mitochondrial matrix, while CoQ is a small, mobile carrier that transfers electrons between the primary dehydrogenases and cytochrome b. CoQ is also restricted to the membrane phase because of its hydrophobic character. The mitochondrial electron transport proteins are clustered into complexes (as shown above) known as Complexes I, II, III, and IV. Complex I, also known as NADH:CoQ oxidoreductase, is composed of NADH dehydrogenase with FMN as cofactor, plus non-heme-iron proteins having at least 1 iron sulfur center. Complex I is responsible for transferring electrons from NADH to CoQ. The ∆E0

' for the latter transfer is 0.42 V ,corresponding to a ∆G' of -19 kcal/mol of electrons transferred. With its highly exergonic free energy change, the flow of electrons through Complex I is more than adequate to drive ATP synthesis. Complex II is also known as succinate dehydrogenase or succinate:CoQ oxidoreductase. The ∆E0

' for electron flow through Complex II is about 0.05 V, corresponding to a ∆G' of -2.3 kcal/mol of electrons transferred, which is insufficient to drive ATP synthesis. The difference in free energy of electron flow through Complexes I and II accounts for the fact that a pair of electrons originating from NADH and passing to oxygen supports production of 3 equivalents of ATP, while 2 electrons from succinate support the production of only 2 equivalents of ATP. Reduced CoQ (CoQH2) diffuses in the lipid phase of the membrane and donates its electrons to Complex III, whose principal components are the heme proteins known as cytochromes b and c1 and a non-heme-iron protein, known as the Rieske iron sulfur protein. In contrast to the heme of hemoglobin and myoglobin, the heme iron of all cytochromes participates in the cyclic redox reactions of electron transport, alternating between the oxidized (Fe+3) and reduced (Fe+2) forms. The electron carrier from Complex III to Complex IV is the smallest of the cytochromes, cytochrome c (molecular weight 12,000). Complex IV, also known as cytochrome oxidase, contains the hemeproteins known as cytochrome a and cytochrome a3, as well as copper-containing proteins in which the copper undergoes a transition from Cu+ to Cu2+ during the transfer of

electrons through the complex to molecular oxygen. Oxygen is the final electron acceptor, with water being the final product of oxygen reduction. Normal oxidation of NADH or succinate is always a 2-electron reaction, with the transfer of 2 hydride ions to a flavin. A hydride ion is composed of 1 proton and 1 electron. Unlike NADH and succinate, flavins can participate in either 1-electron or 2-electron reactions; thus, flavin that is fully reduced by the dehydrogenase reactions can subsequently be oxidized by 2 sequential 1-hydride reactions. The fully reduced form of a flavin is known as the quinol form and the fully oxidized form is known as the quinone form; the intermediate containing a single electron is known as the semiquinone or semiquinol form. Like flavins, CoQ (also known as ubiquinone) can undergo either 1- or 2-electron reactions leading to formation of the reduced quinol, the oxidized quinone, and the semiquinone intermediate. The ability of flavins and CoQ to form semiquinone intermediates is a key feature of the mitochondrial electron transport systems, since these cofactors link the obligatory 2-electron reactions of NADH and succinate with the obligatory 1-electron reactions of the cytochromes. The cytochromes are heme proteins. Like hemoglobin and myoglobin, the cytochromes generally contain 1 heme group per polypeptide---except for cytochrome b, which has 2 heme residues in 1 polypeptide chain. There are 3 forms of heme found in heme proteins, each of which are derived from iron-protoporphyrin IX also called heme b.

Cytochromes vary in the structure of the heme and in its binding to apoprotein. Cytochromes of the c type contain a modified iron protoporphyrin IX known as heme c. In heme c the 2 vinyl (C=C) side chains are covalently bonded to cysteine sulfhydryl residues of the apoprotein. Only cytochromes of the c type contain covalently bound heme. Heme a is also a modified iron protoporphyrin IX. Heme a is found in cytochromes of the a type and in the chlorophyll of green plants.

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Oxidative Phosphorylation

The free energy available as a consequence of transferring 2 electrons from NADH or succinate to molecular oxygen is -57 and -36 kcal/mol, respectively. Oxidative phosphorylation traps this energy as the high-energy phosphate of ATP. In order for oxidative phosphorylation to proceed, two principal conditions must be met. First, the inner mitochondrial membrane must be physically intact so that protons can only reenter the mitochondrion by a process coupled to ATP synthesis. Second, a high concentration of protons must be developed on the outside of the inner membrane. The energy of the proton gradient is known as the chemiosmotic potential, or proton motive force (PMF). This potential is the sum of the concentration difference of protons across the membrane and the difference in electrical charge across the membrane. The 2 electrons from NADH generate a 6-proton gradient. Thus, oxidation of 1 mole of NADH leads to the availability of a PMF with a free energy of about -31.2 kcal (6 x -5.2 kcal). The energy of the gradient is used to drive ATP synthesis as the protons are transported back down their thermodynamic gradient into the mitochondrion. Electrons return to the mitochondrion through the integral membrane protein known as ATP synthase (or Complex V). ATP synthase is a multiple subunit complex that binds ADP and inorganic phosphate at its catalytic site inside the mitochondrion, and requires a proton gradient for activity in the forward direction.

ATP synthase is composed of 3 fragments: F0, which is localized in the membrane; F1, which protrudes from the inside of the inner membrane into the matrix; and oligomycin sensitivity--conferring protein (OSCP), which connects F0 to F1. In damaged mitochondria, permeable to protons, the ATP synthase reaction is active in the reverse direction acting as a very efficient ATP hydrolase or ATPase. back to the top

Stoichiometry of Oxidative Phosphorylation

For each pair of electrons originating from NADH, 3 equivalents of ATP are synthesized, requiring 22.4 kcal of energy. Thus, with 31.2 kcal of available energy, it is clear that the proton gradient generated by electron transport contains sufficient energy to drive normal ATP synthesis. Electrons from succinate have about 2/3 the energy of NADH electrons: they generate PMFs that are about 2/3 as great as NADH electrons and lead to the synthesis of only 2 moles of ATP per mole of succinate oxidized. back to the top

Regulation of Oxidative Phosphorylation

Since electron transport is directly coupled to proton translocation, the flow of electrons through the electron transport system is regulated by the magnitude of the PMF. The higher the PMF the lower the rate of electron transport, and vice versa. Under resting conditions, with a high cell energy charge, the demand for new synthesis of ATP is limited and, although the PMF is high, flow of protons back into the mitochondria through ATP synthase is minimal. When energy demands are increased, such as during vigorous muscle activity, cytosolic ADP rises and is exchanged with intramitochondrial ATP via the transmembrane adenine nucleotide carrier ADP/ATP translocase. Increased intramitochondrial concentrations of ADP cause the PMF to become discharged as protons pour through ATP synthase, regenerating the ATP pool. Thus, while the rate of electron transport is dependent on the PMF, the magnitude of the PMF at any moment simply reflects the energy charge of the cell. In turn the energy charge, or more precisely ADP concentration, normally determines the rate of electron transport by mass action principles. The rate of electron transport is usually measured by assaying the rate of oxygen consumption and is referred to as the cellular respiratory rate. The respiratory rate is known as the state 4 rate when the energy charge is high, the concentration of ADP is low, and electron transport is limited by ADP. When ADP levels rise and inorganic phosphate is available, the flow of protons through ATP synthase is elevated and higher rates of electron transport are observed; the resultant respiratory rate is known as the state 3 rate. Thus, under physiological conditions mitochondrial respiratory activity cycles between state 3 and state 4 rates. back to the top

Inhibitors of Oxidative Phosphorylation

The pathway of electron flow through the electron transport assembly, and the unique properties of the PMF, have been determined through the uses of a number of important antimetabolites. Some of these agents are inhibitors of electron transport at specific sites in the electron transport assembly, while others stimulate electron transport by discharging the proton gradient. For example, antimycin A is a specific inhibitor of cytochrome b. In the presence of antimycin A, cytochrome b can be reduced but not oxidized. As expected, in the presence of cytochrome c remains oxidized in the presence of antimycin A, as do the downstream cytochromes a and a3. An important class of antimetabolites are the uncoupling agents exemplified by 2,4-dinitrophenol (DNP). Uncoupling agents act as lipophilic weak acids, associating with protons on the exterior of mitochondria, passing through the membrane with the bound proton, and dissociating the proton on the interior of the mitochondrion. These agents cause maximum respiratory rates but the electron transport generates no ATP, since the translocated protons do not return to the interior through ATP synthase.

Inhibitors of Oxidative Phosphorylation

Name Function Site of Action

Rotenone e- transport inhibitor Complex I

Amytal e- transport inhibitor Complex I

Antimycin A e- transport inhibitor Complex III

Cyanide e- transport inhibitor Complex IV

Carbon Monoxide e- transport inhibitor Complex IV

Azide e- transport inhibitor Complex IV

2,4,-dinitrophenol Uncoupling agent transmembrane H+ carrier

Pentachlorophenol Uncoupling agent transmembrane H+ carrier

Oligomycin Inhibits ATP synthase

OSCP fraction of ATP synthase

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Energy from Cytosolic NADH

In contrast to oxidation of mitochondrial NADH, cytosolic NADH when oxidized via the electron transport system gives rise to 2 equivalents of ATP if it is

oxidized by the glycerol phosphate shuttle and 3 ATPs if it proceeds via the malate aspartate shuttle. The glycerol phosphate shuttle is coupled to an inner mitochondrial membrane, FAD-linked dehydrogenase, of low energy potential

like that found in Complex II. Thus, cytosolic NADH oxidized by this pathway can generate only 2 equivalents of ATP. The shuttle involves two different glycerol-3-

phosphate dehydrogenases: one is cytosolic, acting to produce glycerol-3-phosphate, and one is an integral protein of the inner mitochondrial membrane that acts to oxidize the glycerol-3-phosphate produced by the cytosolic enzyme. The net result of the process is that reducing equivalents from cytosolic NADH are transferred to the mitochondrial electron transport system. The catalytic site

of the mitochondrial glycerol phosphate dehydrogenase is on the outer surface of the inner membrane, allowing ready access to the product of the second, or

cytosolic, glycerol-3-phosphate dehydrogenase. In some tissues, such as that of heart and muscle, mitochondrial glycerol-3-phosphate dehydrogenase is present in very low amounts, and the malate aspartate shuttle is the dominant pathway for aerobic oxidation of cytosolic

NADH. In contrast to the glycerol phosphate shuttle, the malate aspartate shuttle generates 3 equivalents of ATP for every cytosolic NADH oxidized.

In action, NADH efficiently reduces oxaloacetate (OAA) to malate via cytosolic malate dehydrogenase (MDH) . Malate is transported to the interior of the

mitochondrion via the α-ketoglutarate/malate antiporter. Inside the mitochondrion, malate is oxidized by the MDH of the TCA cycle, producing OAA

and NADH. In this step the cytosolic, NADH-derived reducing equivalents become available to the NADH dehydrogenase of the inner mitochondrial membrane and are oxidized, giving rise to 3 ATPs as described earlier. The

mitochondrial transaminase uses glutamate to convert membrane-impermeable OAA to aspartate and α-ketoglutarate. This provides a pool of α-ketoglutarate for

the aforementioned antiporter. The aspartate which is also produced is translocated out of the mitochondrion.

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Other Biological Oxidations Oxidase complexes, like cytochrome oxidase, transfer electrons directly from NADH and other substrates to oxygen, producing water. Oxygenases, widely localized in membranes of the endoplasmic reticulum, catalyze the addition of

molecular oxygen to organic molecules. There are 2 kinds of oxygenase complexes, monooxygenases and dioxygenases. Dioxygenases add the 2

atoms of molecular oxygen (O2) to carbon and nitrogen of organic compounds. Monooxygenase complexes play a key role in detoxifying drugs and other

compounds (e.g., PCBs and dioxin) and in the normal metabolism of steroids, fatty acids and fat soluble vitamins. Monooxygenases act by sequentially

transferring 2 electrons from NADH or NADPH to 1 of the 2 atoms of oxygen in O2, generating H2O from 1 oxygen atom and incorporating the other oxygen atom

into an organic compound as a hydroxyl group (R-OH). The hydroxylated

products are markedly more water-soluble than their precursors and are much more readily excreted from the body. Widely used synonyms for the

monooxygenases are: mixed function oxidases, hydroxylases, and mixed function hydroxylases.

The chief components of monooxygenase complexes include cytochrome b5, cytochrome P450, and cytochrome P450 reductase, which contains FAD plus

FMN. There are many P450 isozymes; for example, up to 50 different P450 gene products can be found in liver, where the bulk of drug metabolism occurs. Some

of these same gene products are also found in other tissues, where they are responsible for tissue-specific oxygenase activities. P450 reducing equivalents

arise either from NADH via cytochrome b5 or from NADPH via cytochrome P450 reductase, both of which are associated with cytochrome P450 in the

membrane-localized complexes. Enzymatic reactions involving molecular oxygen usually produce water or organic

oxygen in well regulated reactions having specific products. However, under some metabolic conditions (e.g., reperfusion of anaerobic tissues) unpaired electrons gain access to molecular oxygen in unregulated, non-enzymatic

reactions. The products, called free radicals, are quite toxic. These free radicals, especially hydroxy radical, randomly attack all cell components, including

proteins, lipids and nucleic acids, potentially causing extensive cellular damage. Tissues are replete with enzymes to protect against the random chemical reactions that these free radicals initiate. Several free radical scavenging

enzymes have been identified. Superoxide dismutases (SODs) in animals contain either zinc (Zn2+) and

copper (Cu2+), known as CuZnSOD, or manganese (Mn2+) as in the case of the mitochondrial form. These SODs convert superoxide to peroxide and thereby minimizes production of hydroxy radical, the most potent of the oxygen free radicals. Peroxides produced by SOD are also toxic. They are detoxified by

conversion to water via the enzyme peroxidase. The best known mammalian peroxidase is glutathione peroxidase, which contains the modified amino acid

selenocysteine in its reactive center. Glutathione (see the Pentose Phosphate Page) is important in maintaining the normal reduction potential of cells and provides the reducing equivalents for

glutathione peroxidase to convert hydrogen peroxide to water. In red blood cells the lack of glutathione leads to extensive peroxide attack on the plasma

membrane, producing fragile red blood cells that readily undergo hemolysis. Catalase (located in peroxisomes) provides a reductant route for the degradation of hydrogen peroxide. Mammalian catalase has the highest turnover number of

any documented enzyme. back to the top



Last modified: Monday, 29-Sep-2003 11:08:50 EST

• Introduction • Mobilization of Fat Stores • Oxidation Reactions • Alternative Oxidation Pathways • Regulation of Fatty Acid Metabolism • Clinical Aspects of Fatty Acid Metabolism • Ketogenesis • Regulation of Ketogenesis • Clinical Significance of Ketogenesis


Introduction

Utilization of dietary lipids requires that they first be absorbed through the intestine. As these molecules are oils they would be essentially insoluble in the aqueous intestinal environment. Solubilization (emulsification) of dietary lipid is accomplished via bile salts that are synthesized in the liver and secreted from the gallbladder. The emulsified fats can then be degraded by pancreatic lipases (lipase and phospholipase A2). These enzymes, secreted into the intestine from the pancreas, generate free fatty acids and a mixtures of mono- and diacylglycerols from dietary triacylglycerols. Pancreatic lipase degrades triacylglycerols at the 1 and 3 positions sequentially to generate 1,2-diacylglycerols and 2-acylglycerols. Phospholipids are degraded at the 2 position by pancreatic phospholipase A2 releasing a free fatty acid and the lysophospholipid. Following absorption of the products of pancreatic lipase by the intestinal mucosal cells, the resynthesis of triacylglycerols occurs. The triacylglycerols are then solubilized in lipoprotein complexes (complexes of lipid and protein) called chylomicrons. A chylomicron contains lipid droplets surrounded by the more polar lipids and finally a layer of proteins. Triacylglycerols synthesized in the liver are packaged into VLDLs and released into the blood directly. Chylomicrons from the intestine are then released into the blood via the lymph system for delivery to the various tissues for storage or production of energy through oxidation. The triacylglycerol components of VLDLs and chylomicrons are hydrolyzed to free fatty acids and glycerol in the capillaries of adipose tissue and skeletal muscle by the action of lipoprotein lipase. The free fatty acids are then absorbed by the cells and the glycerol is returned via the blood to the liver (and kidneys). The glycerol is then converted to the glycolytic intermediate DHAP.

The classification of blood lipids is distinguished based upon the density of the different lipoproteins. As lipid is less dense than protein, the lower the density of lipoprotein the less protein there is. back to the top

Mobilization of Fat Stores

The primary sources of fatty acids for oxidation are dietary and mobilization from cellular stores. Fatty acids from the diet can are delivered from the gut to cells via transport in the blood. Fatty acids are stored in the form of triacylglycerols primarily within adipocytes of adipose tissue. In response to energy demands, the fatty acids of stored triacylglycerols can be mobilized for use by peripheral tissues. The release of metabolic energy, in the form of fatty acids, is controlled by a complex series of interrelated cascades that result in the activation of hormone-sensitive lipase. The stimulus to activate this cascade, in adipocytes, can be glucagon, epinephrine or β-corticotropin. These hormones bind cell-surface receptors that are coupled to the activation of adenylate cyclase upon ligand binding. The resultant increase in cAMP leads to activation of PKA, which in turn phosphorylates and activates hormone-sensitive lipase. This enzyme hydrolyzes fatty acids from carbon atoms 1 or 3 of triacylglycerols. The resulting diacylglycerols are substrates for either hormone-sensitive lipase or for the non-inducible enzyme diacylglycerol lipase. Finally the monoacylglycerols are substrates for monoacylglycerol lipase. The net result of the action of these enzymes is three moles of free fatty acid and one mole of glycerol. The free fatty acids diffuse from adipose cells, combine with albumin in the blood, and are thereby transported to other tissues, where they passively diffuse into cells.

Model for the activation of hormone-sensitive lipase by epinephrine. Epinephrine binds its' receptor and leads to the activation of adenylate cyclase. The resultant increase in cAMP activates PKA which then phosphorylates and activates hormone-sensitive lipase. Hormone-sensitive lipase hydrolyzes fatty acids from triacylglycerols and diacylglycerols. The final fatty acid is released from monoacylglycerols through the action of monoacylglycerol lipase, an enzyme active in the absence of hormonal stimulation.

In contrast to the hormonal activation of adenylate cyclase and (subsequently) hormone-sensitive lipase in adipocytes, the mobilization of fat from adipose tissue is inhibited by numerous stimuli. The most significant inhibition is that exerted upon adenylate cyclase by insulin. When an individual is well fed state, insulin released from the pancreas prevents the inappropriate mobilization of stored fat. Instead, any excess fat and carbohydrate are incorporated into the triacylglycerol pool within adipose tissue. back to the top

Reactions of Oxidation

Fatty acids must be activated in the cytoplasm before being oxidized in the mitochondria. Activation is catalyzed by fatty acyl-CoA ligase (also called acyl-CoA synthetase or thiokinase). The net result of this activation process is the consumption of 2 molar equivalents of ATP.

Fatty acid + ATP + CoA -------> Acyl-CoA + PPi + AMP Oxidation of fatty acids occurs in the mitochondria. The transport of fatty acyl-CoA into the mitochondria is accomplished via an acyl-carnitine intermediate, which itself is generated by the action of carnitine acyltransferase I, an enzyme that resides in the outer mitochondrial membrane. The acyl-carnitine molecule then is transported into the mitochondria where carnitine acyltransferase II catalyzes the regeneration of the fatty acyl-CoA molecule.

Transport of fatty acids from the cytoplasm to the inner mitochondrial space for oxidation. Following activation to a fatty-CoA, the CoA is exchanged for carnitine by carnitine-palmitoyltransferase I. The fatty-carnitine is then transported to the inside of the mitochondrion where a reversal exchange takes place through the action of carnitine-palmitoyltransferase II. Once inside the mitochondrion the fatty-CoA is a substrate for the β-oxidation machinery.

The process of fatty acid oxidation is termed β-oxidation since it occurs through the sequential removal of 2-carbon units by oxidation at the β-carbon position of

the fatty acyl-CoA molecule. Each round of β-oxidation produces one mole of NADH, one mole of FADH2 and one mole of acetyl-CoA. The acetyl-CoA--- the end product of each round of β-

oxidation--- then enters the TCA cycle, where it is further oxidized to CO2 with the concomitant generation of three moles of NADH, one mole of FADH2 and one mole of ATP. The NADH and FADH2 generated during the fat oxidation and

acetyl-CoA oxidation in the TCA cycle then can enter the respiratory pathway for the production of ATP.

The oxidation of fatty acids yields significantly more energy per carbon atom than does the oxidation of carbohydrates. The net result of the oxidation of one mole

of oleic acid (an 18-carbon fatty acid) will be 146 moles of ATP (2 mole equivalents are used during the activation of the fatty acid), as compared with

114 moles from an equivalent number of glucose carbon atoms. back to the top

Alternative Oxidation Pathways

The majority of natural lipids contain an even number of carbon atoms. A small proportion that contain odd numbers; upon complete β-oxidation, these yield acetyl-CoA units plus a single mole of propionyl-CoA. The propionyl-CoA is

converted, in an ATP-dependent pathway, to succinyl-CoA. The succinyl-CoA can then enter the TCA cycle for further oxidation.

The oxidation of unsaturated fatty acids is essentially the same process as for saturated fats, except when a double bond is encountered. In such a case, the

bond is isomerized by a specific enoyl-CoA isomerase and oxidation continues. In the case of linoleate, the presence of the ∆-12 unsaturation results in the

formation of a dienoyl-CoA during oxidation. This molecule is the substrate for an additional oxidizing enzyme, the NADPH requiring 2,4-dienoyl-CoA reductase.

Phytanic acid is a fatty acid present in the tissues of ruminants and in dairy products and is, therefore, an important dietary component of fatty acid intake. Because phytanic acid is methylated, it cannot act as a substrate for the first enzyme of the β-oxidation pathway (acyl-CoA dehydrogenase). An additional

mitochondrial enzyme, αααα-hydroxylase, adds a hydroxyl group to the α-carbon of phytanic acid, which then serves as a substrate for the remainder of the normal

oxidative enzymes. This process is termed α-oxidation. back to the top

Regulation of Fatty Acid Metabolism

In order to understand how the synthesis and degradation of fats needs to be exquisitely regulated, one must consider the energy requirements of the

organism as a whole. The blood is the carrier of triacylglycerols in the form of VLDLs and chylomicrons, fatty acids bound to albumin, amino acids, lactate, ketone bodies and glucose. The pancreas is the primary organ involved in sensing the organism's dietary and energetic states by monitoring glucose concentrations in the blood. Low blood glucose stimulates the secretion of

glucagon, whereas, elevated blood glucose calls for the secretion of insulin. The metabolism of fat is regulated by two distinct mechanisms. One is short-

term regulation, which can come about through events such as substrate availability, allosteric effectors and/or enzyme modification. The other

mechanism, long-term regulation, is achieved by alteration of the rate of enzyme synthesis and turn-over.

ACC is the rate-limiting (committed) step in fatty acid synthesis. This enzyme is activated by citrate and inhibited by palmitoyl-CoA and other long-chain fatty

acyl-CoAs. ACC activity can also be affected by phosphorylation. For instance, glucagon-stimulated increases in PKA activity result in the phosphorylation of

certain serine residues in ACC leading to decreased activity of the enzyme. By contrast, insulin leads to PKA-independent phosphorylation of ACC at sites

distinct from glucagon, which bring about increased ACC activity. Both of these reaction chains are examples of short-term regulation.

Insulin, a product of the well-fed state, stimulates ACC and FAS synthesis, whereas starvation leads to a decrease in the synthesis of these enzymes. Adipose tissue levels of lipoprotein lipase also are increased by insulin and decreased by starvation. However, the effects of insulin and starvation on

lipoprotein lipase in the heart are just the inverse of those in adipose tissue. This sensitivity allows the heart to absorb any available fatty acids in the blood in

order to oxidize them for energy production. Starvation also leads to increases in the levels of cardiac enzymes of fatty acid oxidation, and to decreases in FAS

and related enzymes of synthesis. Adipose tissue contains hormone-sensitive lipase, which is activated by PKA-dependent phosphorylation; this activation increases the release of fatty acids

into the blood. This in turn leads to the increased oxidation of fatty acids in other tissues such as muscle and liver. In the liver, the net result (due to increased acetyl-CoA levels) is the production of ketone bodies (see below). This would occur under conditions in which the carbohydrate stores and gluconeogenic precursors available in the liver are not sufficient to allow increased glucose

production. The increased levels of fatty acid that become available in response to glucagon or epinephrine are assured of being completely oxidized, because PKA also phosphorylates ACC; the synthesis of fatty acid is thereby inhibited. Insulin has the opposite effect to glucagon and epinephrine: it increases the

synthesis of triacylglycerols (and glycogen). One of the many effects of insulin is to lower cAMP levels, which leads to increased dephosphorylation through the enhanced activity of protein phosphatases such as PP-1. With respect to fatty acid metabolism, this yields dephosphorylated and inactive hormone-sensitive

lipase. Insulin also stimulates certain phosphorylation events. This occurs through activation of several cAMP-independent kinases, one of which

phosphorylates and thereby stimulates the activity of ACC. Fat metabolism can also be regulated by malonyl-CoA-mediated inhibition of

carnitine acyltransferase I. Such regulation serves to prevent de novo synthesized fatty acids from entering the mitochondria and being oxidized.

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Clinical Significance of Fatty Acids The majority of clinical problems related to fatty acid metabolism are associated

with processes of oxidation. These disorders fall into four main groups:

• 1. Deficiencies in Carnitine: Deficiencies in carnitine lead to an inability to transport fatty acids into the mitochondria for oxidation. This can occur

in newborns and particularly in pre-term infants. Carnitine deficiencies also are found in patients undergoing hemodialysis or exhibiting organic

aciduria. Carnitine deficiencies may manifest systemic symptomology or may be limited to only muscles. Symptoms can range from mild occasional muscle cramping to severe weakness or even death.

Treatment is by oral carnitine administration. • 2. Carnitine Palmitoyltransferase I (CPT I) Deficiency: Deficiencies in this

enzyme affect primarily the liver and lead to reduced fatty acid oxidation and ketogenesis. Carnitine Palmitoylransferase II (CPT II) deficiency

results in recurrent muscle pain and fatigue and myoglobinuria following strenuous exercise. Carnitine acyltransferases may also be inhibited by

sulfonylurea drugs such as tolbutamide and glyburide. • 3. Deficiencies in Acyl-CoA Dehydrogenases: A group of inherited

diseases that impair β-oxidation result from deficiencies in acyl-CoA dehydrogenases. The enzymes affected may belong to one of four

categories: o very long-chain acyl-CoA dehydrogenase (VLCAD) Disease

description o long-chain acyl-CoA dehydrogenase (LCAD) Disease

description o medium-chain acyl-CoA dehydrogenase (MCAD) Disease

description o short-chain acyl-CoA dehydrogenase (SCAD) Disease

description

MCAD deficiency is the most common form of this disease. In the first years of life this deficiency will become apparent following a prolonged fasting period. Symptoms include vomiting, lethargy and frequently coma. Excessive urinary

excretion of medium-chain dicarboxylic acids as well as their glycine and carnitine esters is diagnostic of this condition. In the case of this enzyme

deficiency. taking care to avoid prolonged fasting is sufficient to prevent clinical problems.

• 4. Refsum's Disease: Refsum's disease is a rare inherited disorder in which patients lack the mitochondrial α-oxidizing enzyme. As a

consequence, they accumulate large quantities of phytanic acid in their tissues and serum. This leads to severe symptoms, including cerebellar

ataxia, retinitis pigmentosa, nerve deafness and peripheral neuropathy. As expected, the restriction of dairy products and ruminant meat from the diet

can ameliorate the symptoms of this disease.

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Ketogenesis

During high rates of fatty acid oxidation, primarily in the liver, large amounts of acetyl-CoA are generated. These exceed the capacity of the TCA cycle, and one result is the synthesis of ketone bodies, or ketogenesis. The ketone bodies are

acetoacetate, ββββ-hydroxybutyrate, and acetone. The formation of acetoacetyl-CoA occurs by condensation of two moles of acetyl-

CoA through a reversal of the thiolase catalyzed reaction of fat oxidation. Acetoacetyl-CoA and an additional acetyl-CoA are converted to ββββ-hydroxy-ββββ-

methylglutaryl-CoA (HMG-CoA) by HMG-CoA synthase, an enzyme found in large amounts only in the liver. Some of the HMG-CoA leaves the mitochondria, where it is converted to mevalonate (the precursor for cholesterol synthesis) by

HMG-CoA reductase. HMG-CoA in the mitochondria is converted to acetoacetate by the action of HMG-CoA lyase. Acetoacetate can undergo

spontaneous decarboxylation to acetone, or be enzymatically converted to β-hydroxybutyrate through the action of ββββ-hydroxybutyrate dehydrogenase.

When the level of glycogen in the liver is high the production of β-hydroxybutyrate increases.

When carbohydrate utilization is low or deficient, the level of oxaloacetate will also be low, resulting in a reduced flux through the TCA cycle. This in turn leads

to increased release of ketone bodies from the liver for use as fuel by other tissues. In early stages of starvation, when the last remnants of fat are oxidized,

heart and skeletal muscle will consume primarily ketone bodies to preserve glucose for use by the brain. Acetoacetate and β-hydroxybutyrate, in particular, also serve as major substrates for the biosynthesis of neonatal cerebral lipids. Ketone bodies are utilized by extrahepatic tissues through the conversion of β-hydroxybutyrate to acetoacetate and of acetoacetate to acetoacetyl-CoA. The

first step involves the reversal of the β-hydroxybutyrate dehydrogenase reaction, and the second involves the action (shown below) of acetoacetate:succinyl-

CoA transferase, also called ketoacyl-CoA-transferase. Acetoacetate + Succinyl-CoA <------> Acetoacetyl-CoA + succinate

The latter enzyme is present in all tissues except the liver. Importantly, its absence allows the liver to produce ketone bodies but not to utilize them. This

ensures that extrahepatic tissues have access to ketone bodies as a fuel source during prolonged starvation.

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Regulation of Ketogenesis

The fate of the products of fatty acid metabolism is determined by an individual's physiological status. Ketogenesis takes place primarily in the liver and may by

affected by several factors:

• 1. Control in the release of free fatty acids from adipose tissue directly affects the level of ketogenesis in the liver. This is, of course, substrate-

level regulation.

• 2. Once fats enter the liver, they have two distinct fates. They may be activated to acyl-CoAs and oxidized, or esterified to glycerol in the

production of triacylglycerols. If the liver has sufficient supplies of glycerol-3-phosphate, most of the fats will be turned to the production of

triacylglycerols. • 3. The generation of acetyl-CoA by oxidation of fats can be completely

oxidized in the TCA cycle. Therefore, if the demand for ATP is high the fate of acetyl-CoA is likely to be further oxidation to CO2.

• 4. The level of fat oxidation is regulated hormonally through phosphorylation of ACC, which may activate it (in response to glucagon)

or inhibit it (in the case of insulin).

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Clinical Significance of Ketogenesis

The production of ketone bodies occurs at a relatively low rate during normal feeding and under conditions of normal physiological status. Normal

physiological responses to carbohydrate shortages cause the liver to increase the production of ketone bodies from the acetyl-CoA generated from fatty acid oxidation. This allows the heart and skeletal muscles primarily to use ketone

bodies for energy, thereby preserving the limited glucose for use by the brain. The most significant disruption in the level of ketosis, leading to profound clinical

manifestations, occurs in untreated insulin-dependent diabetes mellitus. This physiological state, diabetic ketoacidosis (DKA), results from a reduced supply of glucose (due to a significant decline in circulating insulin) and a concomitant

increase in fatty acid oxidation (due to a concomitant increase in circulating glucagon). The increased production of acetyl-CoA leads to ketone body

production that exceeds the ability of peripheral tissues to oxidize them. Ketone bodies are relatively strong acids (pKa around 3.5), and their increase lowers the

pH of the blood. This acidification of the blood is dangerous chiefly because it impairs the ability of hemoglobin to bind oxygen.

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